Methods and Test Kits for Determining Male Fertility Status

ABSTRACT

This disclosure provides a method for determining male fertility status. The method comprises determining GM1 localization patterns following induced sperm capacitation, identifying the percentage of various patterns, particularly the ratio of [(AA+APM)/total number of GM1 localization patterns] and determining if the percentage of certain GM1 localization patterns in response to induced capacitation is altered. Based on the change in the percentage of localization patterns of certain patterns in response to induced capacitation, alone or in combination with other sperm attributes, male fertility status can be identified.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/296,420, filed on Feb. 17, 2016, U.S. Provisional Application No.62/322,252, filed Apr. 14, 2016, U.S. Provisional Application No.62/328,890 filed Apr. 28, 2016, each of which is incorporated in itsentirety.

FIELD OF THE DISCLOSURE

This invention relates generally to the field of male fertility and morespecifically to determining male fertility status based on G_(M1)ganglioside distribution patterns following induced sperm capacitation.BACKGROUND OF THE DISCLOSURE

In the US, 10% of couples have medical appointments related toinfertility with 40% of infertility being associated with the male.Globally, this translates to over 73 million infertile couples. Typicalmale reproductive health exams assess sperm number, appearance, andmotility. Unfortunately, half of infertile men have sperm that meetnormal parameters for these descriptive criteria and are only identifiedas having “idiopathic infertility” after repeatedly failing at bothnatural conception and techniques of assisted reproduction such asintra-uterine insemination (IUI). Because each failed cycle inflictsgreat physical, emotional, and financial tolls on couples and it coststhe US healthcare system over $5 billion annually, there is a tremendousneed for a practical test of sperm function. Data on sperm functionwould allow clinicians to direct their patients toward a technology ofassisted reproduction that would give them the best chance to conceive.

Upon entrance into the female tract, sperm are not immediately able tofertilize an egg. Rather, they must undergo a process of functionalmaturation known as “capacitation.” This process relies upon theirability to respond to specific stimuli by having specific changes intheir cell membrane, namely a change in the distribution pattern of theganglioside G_(M1) in response to exposure to stimuli for capacitation.

Various G_(M1) localization patterns have been identified and associatedwith capacitation or non-capacitation. In particular, apical acrosome(AA) G_(M1) localization patterns and acrosomal plasma membrane (APM)G_(M1) localization patterns have been associated with capacitation inbovine and human sperm. Sperm capacitation can be quantitativelyexpressed as a Cap-Score™ value, generated via the Cap-Score™ SpermFunction Test (“Cap-Score™ Test” or “Cap-Score”), is defined as ([numberof apical acrosome (AA) G_(M1) localization patterns+number of acrosomalplasma membrane (APM) G_(M1) localization patterns]/total number ofG_(M1) labeled localization patterns) where the number of eachlocalization pattern is measured and then ultimately converted to apercentage score. In addition to APM G_(M1) localization patterns and AAG_(M1) localization patterns, the other labeled localization patternsincluded Lined-Cell G_(M1) localization patterns, intermediate (INTER)G_(M1) localization patterns, post acrosomal plasma membrane (PAPM)G_(M1) localization patterns, apical acrosome/post acrosome (AA/PA)G_(M1) localization patterns, equatorial segment (ES) G_(M1)localization patterns, and diffuse (DIFF) G_(M1) localization patterns.(Travis et al., “Impacts of common semen handling methods on spermfunction,” The Journal of Urology, 195 (4), e909 (2016)).

SUMMARY OF THE DISCLOSURE

In one embodiment, disclosed herein are methods and kits for determiningmale fertility status. In one embodiment, this disclosure describes amethod for identifying male fertility status based on a change in thenumber of certain G_(M1) localization patterns in response to induced invitro capacitation. In one embodiment, the G_(M1) localization patternis a Lined-Cell G_(M1) localization pattern.

An embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method comprising the followingsteps. A sample of in vitro capacitated sperm cells is treated with afluorescence label. One or more capacitated-fluorescence images isobtained wherein the images display one or more G_(M1) localizationpatterns associated with fluorescence labeled in vitro capacitated spermcells. An apical acrosome (AA) G_(M1) localization pattern and anacrosomal plasma membrane (APM) G_(M1) localization pattern are eachassigned to a capacitated state and a Lined-Cell G_(M1) localizationpattern and all other labeled G_(M1) localization patterns are assignedto a non-capacitated state each displayed in thecapacitated-fluorescence images. A number for G_(M1) localizationpatterns is measured, the patterns comprising AA G_(M1) localizationpattern, APM G_(M1) localization pattern, Lined-Cell G_(M1) localizationpattern and all other labeled G_(M1) localization patterns, for thefluorescence labeled in vitro capacitated sperm cells, displayed in thecapacitated-fluorescence images to determine a percentage of [(AA G_(M1)localization patterns plus APM G_(M1) localization patterns)/totalG_(M1) localization patterns]. In one embodiment, a fertility thresholdassociated with a percentage of [(AA G_(M1) localization patterns plusAPM G_(M1) localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage than onestandard deviation below the reference mean percentage indicatesfertile; less than a percentage that is one standard deviation below thereference mean percentage and greater than a percentage that is twostandard deviations below the reference mean percentage indicatessub-fertile; less than a percentage that is two standard deviationsbelow the reference mean percentage indicates infertile. The percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] for the labeled fixed invitro capacitated sperm sample is compared to the reference percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns]. The fertility thresholdis identified based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

Another embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes exposing afirst portion of a sperm sample from a male to non-capacitatingconditions to obtain an in vitro non-capacitated sperm sample; exposinga second portion of the sperm sample to capacitating conditions toobtain an in vitro capacitated sperm sample; fixing the in vitronon-capacitated sperm sample and the in vitro capacitated sperm samplewith a fixative for a time period of at least: one hour, two hours, fourhours, eight hours, twelve hours, eighteen hours or twenty four hours;treating the fixed in vitro non-capacitated sperm sample and the fixedin vitro capacitated sperm sample with a labeling molecule for G_(M1)localization patterns, wherein the labeling molecule has a detectablelabel; identifying more than one labeled G_(M1) localization patternsfor the labeled fixed in vitro non-capacitated sperm sample and thelabeled fixed in vitro capacitated sperm sample, said G_(M1) labeledlocalization patterns being an apical acrosome (AA) G_(M1) localizationpattern, an acrosomal plasma membrane (APM) G_(M1) localization pattern,a Lined-Cell G_(M1) localization pattern and all other labeled G_(M1)localization patterns; comparing the labeled G_(M1) localizationpatterns for the labeled fixed in vitro non-capacitated sperm sample tothe labeled G_(M1) localization patterns for the labeled fixed in vitrocapacitated sperm sample; based on the comparison, assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male based on the identifiedG_(M1) labeled localization patterns for the labeled fixed in vitronon-capacitated sperm sample and the labeled fixed in vitro capacitatedsperm sample. In one embodiment, the characterizing step comprises thesteps of: determining a fertility threshold associated with a percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] for the labeled fixed invitro capacitated sperm sample; wherein a reference percentage of [(AAG_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns], based on distributionstatistics of a known fertile population corresponding to: greater thana percentage that is one standard deviation below the reference meanpercentage indicates fertile; less than a percentage that is onestandard deviation below the reference mean percentage and greater thana percentage that is two standard deviations below the reference meanpercentage indicates sub-fertile; greater than two standard deviationsbelow the reference mean percentage indicates infertile. The percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] for the labeled fixed invitro capacitated sperm sample is compared to the reference percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns]. The fertility thresholdis identified based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In one embodiment, prior to the exposing steps, a semen sample istreated to decrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembranes chosen from the various reagents that are used to decreasesemen viscosity. In some such embodiments, the membrane damagingreagents potentially may include (i) a protease; (ii) a nuclease (iii) amucolytic agent; (iv) a lipase; (v) an esterase and (vi) glycosidehydrolases. In some embodiments, the identifying step is repeated untilthe number of Lined-Cell G_(M1) localization patterns is substantiallyconstant. In one such embodiment, after the identifying step isperformed, determining the number of Lined-Cell G_(M1) localizationpatterns, for the labeled fixed in vitro capacitated sperm until thenumber is less than 5%, less than 3% of the total number of labeledcells; or ranges from 1% to 5%, 2 to 5% of the total number of labeledcells. In another such embodiment, after the identifying step isperformed, the number of Lined-Cell G_(M1) localization patterns, forthe labeled fixed in vitro non-capacitated sperm is determined until thenumber is less than: 25%, 20%, 15% or 10% of the total number of labeledcells; or ranges from 2% to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5%of the total number of labeled cells. In some embodiments the wideorifice pipette has a gauge size of at least 18 gauge, 16 gauge or 14gauge. In some embodiments, the wide orifice pipette has an orifice sizeof at least 1 mm, 1.2 mm or 1.4 mm.

In another such embodiment, the characterizing step further includes thesteps of: determining the number of each G_(M1) labeled localizationpatterns for a predetermined number of the labeled fixed in vitronon-capacitated sperm sample; determining the number of each G_(M1)labeled localization patterns for a predetermined number of the labeledfixed in vitro capacitated sperm sample; calculating a ratio for a sumof the number of AA G_(M1) localization patterns and number of APMG_(M1) localization patterns over a sum of the total number of G_(M1)labeled localization patterns for the labeled fixed in vitronon-capacitated sperm sample; and calculating a ratio for a sum of thenumber of AA G_(M1) localization patterns and number of APM G_(M1)localization patterns over a sum of the total number of G_(M1) labeledlocalization patterns for the labeled fixed in vitro capacitated spermsample.

In one embodiment disclosed herein the method further includes the stepsof: comparing the ratio for the labeled fixed in vitro non-capacitatedsperm to a ratio of labeled fixed in vitro non-capacitated sperm havinga known fertility status; and comparing the ratio for the labeled fixedin vitro capacitated sperm to a ratio of labeled fixed in vitrocapacitated sperm having a known fertility status.

Yet another embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:obtaining a first portion of a sperm sample from a male that has beenexposed to in vitro non-capacitating conditions, fixed in a fixative forat least: one hour, two hours, four hours, eight hours, twelve hours,eighteen hours or twenty four hours, and treated with a labelingmolecule for G_(M1) localization patterns, wherein the labeling moleculehas a detectable label; obtaining a second portion of the sperm samplethat has been exposed to in vitro capacitating conditions, fixed in afixative, and treated with the labeling molecule for G_(M1) localizationpatterns; identifying more than one G_(M1) labeled localization patternsfor the labeled fixed in vitro non-capacitated sperm sample and thelabeled fixed in vitro capacitated sperm sample, said G_(M1) labeledlocalization patterns being an apical acrosome (AA) G_(M1) localizationpattern, an acrosomal plasma membrane (APM) G_(M1) localization pattern,a Lined-Cell G_(M1) localization pattern and all other labeled G_(M1)localization patterns; comparing the labeled G_(M1) localizationpatterns for the labeled fixed in vitro non-capacitated sperm sample tothe labeled G_(M1) localization patterns for the labeled fixed in vitrocapacitated sperm sample; based on the comparison, assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male based on the identifiedG_(M1) labeled localization patterns for the labeled fixed in vitronon-capacitated sperm sample and the labeled fixed in vitro capacitatedsperm sample. In one such embodiment, the characterizing step comprisesthe steps of: determining a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] for thelabeled fixed in vitro capacitated sperm sample; wherein a referencepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns], based ondistribution statistics of a known fertile population corresponding to:greater than a percentage that is one standard deviation below thereference mean percentage indicates fertile; less than a percentage thatis one standard deviation below the reference mean percentage andgreater than a percentage that is two standard deviations below thereference mean percentage indicates sub-fertile; less than a percentagethat is two standard deviations below the reference mean percentageindicates infertile; comparing the percentage of [(AA G_(M1)localization patterns plus APM G_(M1) localization patterns)/totalG_(M1) localization patterns] for the labeled fixed in vitro capacitatedsperm sample to the reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] and identifying the fertility threshold based onthe comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormalfertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In some embodiments, the identifying step is repeated until the numberof Lined-Cell G_(M1) localization patterns is substantially constant. Inone such embodiment, after the identifying step is performed,determining the number of Lined-Cell G_(M1) localization patterns, forthe labeled fixed in vitro capacitated sperm until the number is lessthan 5%, less than 3% of the total number of labeled cells; or rangesfrom 1% to 5%, 2 to 5% of the total number of labeled cells. In anothersuch embodiment, after the identifying step is performed, determiningthe number of Lined-Cell G_(M1) localization patterns, for the labeledfixed in vitro non-capacitated sperm until the number is less than: 25%,20%, 15% or 10% of the total number of labeled cells; or ranges from 2%to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5% of the total number oflabeled cells.

In one embodiment of such method, the method further includes the stepsof: determining the number of each G_(M1) labeled localization patternsfor a predetermined number of the labeled fixed in vitro non-capacitatedsperm sample and the labeled fixed in vitro capacitated sperm sample,and calculating a ratio for a sum of the number of AA G_(M1)localization patterns and number of APM G_(M1) localization patternsover a sum of the total number of G_(M1) localization patterns each forthe labeled fixed in vitro non-capacitated sperm sample and the labeledfixed in vitro capacitated sperm sample.

In another embodiment, the characterizing step further includes thesteps of: comparing the ratio for the labeled fixed in vitro capacitatedsperm sample to ratios of G_(M1) localization patterns of in vitrocapacitated sperm for males having a known fertility status; andcomparing the ratio for the labeled fixed in vitro non-capacitated spermsample to ratios of G_(M1) localization patterns in vitronon-capacitated sperm for males having a known fertility status.

Still yet another embodiment disclosed herein is a method fordetermining male fertility status. In one embodiment, the methodincludes the steps of: exposing, in vitro, a sperm sample from a male tocapacitating conditions; fixing the capacitated sperm sample with afixative for at least: one hour, two hours, four hours, eight hours,twelve hours, eighteen hours or twenty four hours; treating the fixed invitro capacitated sperm sample with a labeling molecule for G_(M1)localization patterns, wherein the labeling molecule has a detectablelabel; identifying more than one G_(M1) labeled localization patternsfor the labeled fixed in vitro capacitated sperm sample, said G_(M1)labeled localization patterns being an apical acrosome (AA) G_(M1)localization pattern, an acrosomal plasma membrane (APM) G_(M1)localization pattern, a Lined-Cell G_(M1) localization pattern and allother labeled G_(M1) localization patterns; assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male. In one embodiment, thecharacterizing step comprises the steps of: determining a fertilitythreshold associated with a percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] for the labeled fixed in vitro capacitated spermsample; wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesfertile; less than a percentage that is one standard deviation below thereference mean percentage and greater than a percentage that is twostandard deviations below the reference mean percentage indicatessub-fertile; less than two standard deviations below the reference meanpercentage indicates infertile; comparing the percentage of [(AA G_(M1)localization patterns plus APM G_(M1) localization patterns)/totalG_(M1) localization patterns] for the labeled fixed in vitro capacitatedsperm sample to the reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] and identifying the fertility threshold based onthe comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In one embodiment, prior to the exposing steps, a semen sample istreated to decrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembrane chosen from the various reagents that are used to decreasesemen viscosity. In some embodiments, the membrane damaging reagent maybe potentially selected from the group consisting of (i) a protease;(ii) a nuclease (iii) a mucolytic agent; (iv) a lipase; (v) an esteraseand (vi) glycoside hydrolases. In some embodiments, the identifying stepis repeated until the number of Lined-Cell G_(M1) localization patternsis substantially constant. In one such embodiment, after the identifyingstep is performed, determining the number of Lined-Cell G_(M1)localization patterns, for the labeled fixed in vitro capacitated spermuntil the number is less than 5%, less than 3% of the total number oflabeled cells; or ranges from 1% to 5%, 2 to 5% of the total number oflabeled cells. In another such embodiment, after the identifying step isperformed, determining the number of Lined-Cell G_(M1) localizationpatterns, for the labeled fixed in vitro non-capacitated sperm until thenumber is less than: 25%, 20%, 15% or 10% of the total number of labeledcells; or ranges from 2% to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5%of the total number of labeled cells. In some embodiments the wideorifice pipette has a gauge size of at least 18 gauge, 16 gauge or 14gauge. In some embodiments, the wide orifice pipette has an orifice sizeof at least 1 mm, 1.2 mm or 1.4 mm.

In one embodiment of such method, the method further includes the stepsof: comparing the ratio of G_(M1) localization patterns to ratios ofG_(M1) localization patterns for males having a known fertility status.In one such embodiment, the comparing step includes the steps of:determining the number of each G_(M1) labeled localization patterns fora predetermined number of the labeled fixed in vitro capacitated spermsample, and calculating a ratio for a sum of the number of AA G_(M1)localization patterns and number of APM G_(M1) localization patternsover a sum of the total number of G_(M1) labeled localization patterns.

Another embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:obtaining a first portion of a sperm sample from a male that has beenexposed to in vitro capacitating conditions, fixed in a fixative for atleast: one hour, two hours, four hours, eight hours, twelve hours,eighteen hours or twenty four hours, and stained with a labelingmolecule for G_(M1) localization patterns, wherein the labeling moleculehas a detectable label; identifying more than one G_(M1) labeledlocalization patterns for the labeled fixed in vitro capacitated spermsample, said G_(M1) localization patterns being an apical acrosome (AA)G_(M1) localization pattern, an acrosomal plasma membrane (APM) G_(M1)localization pattern, a Lined-Cell G_(M1) localization pattern and allother labeled G_(M1) localization patterns; assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male. In some embodiments,characterizing step comprises the steps of: determining a fertilitythreshold associated with a percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] for the labeled fixed in vitro capacitated spermsample; wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesfertile; less than a percentage that is one standard deviation below thereference mean percentage and greater than a percentage that is twostandard deviations below the reference mean percentage indicatessub-fertile; less than a percentage that is two standard deviationsbelow the reference mean percentage indicates infertile; comparing thepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] for thelabeled fixed in vitro capacitated sperm sample to the referencepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] andidentifying the fertility threshold based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In some embodiments, the identifying step is repeated until the numberof Lined-Cell G_(M1) localization patterns is substantially constant. Inone such embodiment, after the identifying step is performed,determining the number of Lined-Cell G_(M1) localization patterns, forthe labeled fixed in vitro capacitated sperm until the number is lessthan 5%, less than 3% of the total number of labeled cells; or rangesfrom 1% to 5%, 2 to 5% of the total number of labeled cells. In anothersuch embodiment, after the identifying step is performed, determiningthe number of Lined-Cell G_(M1) localization patterns, for the labeledfixed in vitro non-capacitated sperm until the number is less than: 25%,20%, 15% or 10% of the total number of labeled cells; or ranges from 2%to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5% of the total number oflabeled cells.

In one embodiment of such method, the method further includes the stepsof: comparing the ratio of G_(M1) localization patterns to ratios ofG_(M1) localization patterns for males having a known fertility status.In one such embodiment, the comparing step includes the steps of:determining the number of each G_(M1) labeled localization patterns fora predetermined number of the labeled fixed in vitro capacitated spermsample, and calculating a ratio for a sum of the number of AA G_(M1)localization patterns and number of APM G_(M1) localization patternsover a sum of the total number of G_(M1) labeled localization patterns.

Another embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:obtaining a sperm sample, wherein at least a portion of the sperm samplehas been exposed to in vitro capacitating conditions to obtain in vitrocapacitated sperm, has been exposed to a fixative for at least: onehour, two hours, four hours, eight hours, twelve hours, eighteen hoursor twenty four hours, and has been stained for G_(M1); obtaining valuesfor one or more semen parameters of the sperm sample; determining aCap-Score of the labeled fixed in vitro capacitated sperm sample basedon one or more G_(M1) labeled localization patterns, said G_(M1) labeledlocalization patterns being an apical acrosome (AA) G_(M1) localizationpattern, a post-acrosomal plasma membrane (APM) G_(M1) localizationpattern, a Lined-Cell G_(M1) localization pattern and all other labeledG_(M1) localization patterns; and calculating a male fertility index(MFI) value of the male based on the determined CAP score and the one ormore obtained semen parameters. In one embodiment, the one or more semenparameters of the sperm sample are selected from the group consisting ofvolume of the original sperm sample, concentration of sperm, motility ofsperm, and morphology of sperm. In some embodiments, the identifyingstep is repeated until the number of Lined-Cell G_(M1) localizationpatterns is substantially constant.

In one such embodiment, after the identifying step is performed,determining the number of Lined-Cell G_(M1) localization patterns, forthe labeled fixed in vitro capacitated sperm until the number is lessthan 5%, less than 3% of the total number of labeled cells; or rangesfrom 1% to 5%, 2 to 5% of the total number of labeled cells. In anothersuch embodiment, after the identifying step is performed, determiningthe number of Lined-Cell G_(M1) localization patterns, for the labeledfixed in vitro non-capacitated sperm until the number is less than: 25%,20%, 15% or 10% of the total number of labeled cells; or ranges from 2%to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5% of the total number oflabeled cells.

In various embodiments of the methods described herein, the more thanone of G_(M1) labeled localization patterns comprises AA G_(M1)localization pattern, APM G_(M1) localization pattern, Lined-Cell G_(M1)localization pattern, intermediate (INTER) G_(M1) localization pattern,post acrosomal plasma membrane (PAPM) G_(M1) localization pattern,apical acrosome/post acrosome (AA/PA) G_(M1) localization pattern,equatorial segment (ES) G_(M1) localization pattern, and diffuse (DIFF)G_(M1) localization pattern.

In one embodiment, exposing the first portion of the sperm sample tonon-capacitating conditions and exposing the second portion of the spermsample to capacitating conditions occur concurrently.

In one embodiment disclosed herein is a kit for identifying a fertilitystatus of a male comprising: a diagram illustrating one or more G_(M1)localization patterns of capacitated sperm and one of more G_(M1)localization patterns of non-capacitated sperm, wherein said G_(M1)localization patterns of capacitated sperm and G_(M1) localizationpatterns of non-capacitated sperm are reflective of known fertilitystatus; a wide orifice pipette having an orifice of sufficient size indiameter to prevent shearing of a sperm membrane; one or more of thefollowing: capacitating media, non-capacitating media, fixativecomposition, labeling reagents for determining G_(M1) localizationpatterns; with the proviso that the fixative composition does not damagesperm membranes, wherein the capacitating media and non-capacitatingmedia, when applied in vitro to sperm cells, produce G_(M1) localizationpatterns indicative of capacitated sperm and patterns indicative ofnon-capacitated sperm as reflected in the diagram. In one embodiment,the kit contains instructions for handling sperm in order to avoiddamaging the sperm membrane. In some embodiments the wide orificepipette has a gauge size of at least 18 gauge, 16 gauge or 14 gauge. Insome embodiments, the wide orifice pipette has an orifice size of atleast 1 mm, 1.2 mm or 1.4 mm.

In certain embodiments described herein, the in vitro capacitatingconditions include exposure to one or more of bicarbonate ions, calciumions, and a mediator of sterol efflux. In some embodiments, the mediatorof sterol efflux is 2-hydroxy-propyl-β-cyclodextrin,methyl-β-cyclodextrin, serum albumin, high density lipoprotein,phospholipid vesicles, fetal cord serum ultrafiltrate, fatty acidbinding proteins, or liposomes. In one embodiment, the mediator ofsterol efflux is 2-hydroxy-propyl-β-cyclodextrin.

In one embodiment, the non-capacitating conditions include the lack ofexposure to one or more of bicarbonate ions, calcium ions, and amediator of sterol efflux.

In certain embodiments described herein, the fixative is an aldehydefixative. In one embodiment, the fixative includes paraformaldehyde,glutaraldehyde or combinations thereof. In certain embodiments, theaffinity molecule for G_(M1) is fluorescent labeled cholera toxin bsubunit.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofembodiments of the methods and kits for determining male fertilitystatus, will be better understood when read in conjunction with theappended drawings. It should be understood, however, that the inventionis not limited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 shows INTER, APM, AA, PAPM, AA/PA, ES, and DIFF localizationpatterns of G_(M1) in normal human sperm and sperm from infertile malesunder non-capacitating conditions or capacitating conditions;

FIG. 2A shows the relative distributions of the INTER, APM, AA, PAPM,AA/PA, ES, and DIFF localization patterns of G_(M1) in normal humansperm under non-capacitating conditions;

FIG. 2B shows the relative distributions of the INTER, APM, AA, PAPM,AA/PA, ES, and DIFF localization patterns of G_(M1) in normal humansperm under capacitating conditions;

FIG. 2C shows the relative distributions of the INTER, APM, AA, PAPM,AA/PA, ES, and DIFF localization patterns of G_(M1) in human sperm frominfertile males under capacitating conditions;

FIG. 3 shows the relative number of the combined APM and AAlocalizations patterns as a function of time of incubation in humansperm for a group normal males and in human sperm for a group infertilemales, under capacitating conditions and non-capacitating conditions,and the clinical outcomes for each group of males;

FIG. 4 shows the percentage of AA and APM localization patterns in spermfrom known fertile donors incubated with stimuli promoting capacitation;

FIG. 5 shows a comparison of the percentage of AA and APM localizationpatterns in sperm from suspected sub-fertile/infertile donors with thestatistical thresholds of fertile men; and

FIGS. 6A, 6B, 6C, and 6D show Lined-Cell G_(M1) localization patterns ofG_(M1) in sperm from infertile males under capacitating conditions.

DETAILED DESCRIPTION OF THE DISCLOSURE

With reference to the accompanying drawings, various embodiments of thepresent invention are described more fully below. Some but not allembodiments of the present invention are shown. Indeed, variousembodiments of the invention may be embodied in many different forms andshould not be construed as limited to the embodiments expresslydescribed. It is to be understood that at least some of the figures anddescriptions of the invention have been simplified to focus on elementsthat are relevant for a clear understanding of the invention, whileeliminating, for purposes of clarity, other elements that those ofordinary skill in the art will appreciate may also comprise a portion ofthe invention. However, because such elements are well known in the art,and because they do not necessarily facilitate a better understanding ofthe invention, a description of such elements is not provided herein.

Each and every reference identified herein is incorporated by referencein its entirety.

Unless specifically set forth herein, the terms “a”, “an” and “the” arenot limited to one element but instead should be read as meaning “atleast one”.

“About” is understood to mean the range of + and −10% of the valuereferenced. However, use of “about” in reference to a value does notexclude the possibility of the referenced value alone. For example,“about 1 hour” is understood to fully support “54 minutes,” “1 hour,”and “66 minutes.”

The present disclosure is based on the observations that certain G_(M1)localization patterns can provide information regarding male fertilitystatus. Determination of G_(M1) localization patterns is described inU.S. Pat. Nos. 7,160,676, 7,670,763, and 8,367,313, the disclosures ofwhich are incorporated herein by reference. This disclosure providesmethods and kits for determination of male fertility status. In certainembodiments, the method is based on a change in the percentage ofcertain G_(M1) localization patterns upon exposure to in vitrocapacitating stimuli. In other embodiments, the method is basedspecifically on a change in the percentage of a Lined-Cell G_(M1)localization pattern upon exposure to in vitro capacitating stimuli.

In one embodiment, disclosed herein is a method for determining malefertility status. In one embodiment, the method includes subjecting asperm sample from an individual to in vitro capacitating and in vitronon-capacitating conditions, determining a change in the percentage ofcertain G_(M1) localization patterns upon exposure to in vitrocapacitating conditions, and based on the level of change, identifyingthe fertility status.

The term “in vitro capacitated” sperm refers to sperm which have beenincubated under conditions which promote the process of capacitation. Inone embodiment, capacitation conditions include the presence in themedium of one or more of bicarbonate ions, calcium ions, and a sterolacceptor, e.g., serum albumin or a cyclodextrin. In one embodiment, invitro capacitation conditions include the presence of bicarbonate andcalcium ions in the medium, and the presence of a sterol acceptor. Inone embodiment, a sterol acceptor is a mediator of sterol efflux.Capacitated sperm have acquired the ability to undergo acrosomeexocytosis and have acquired a hyperactivated pattern of motility. Theterm “in vitro non-capacitated” sperm refers to sperm which are notincubated with one or more of the above-listed stimuli for capacitation.In one embodiment, non-capacitation conditions include the absence ofcapacitation conditions. In another embodiment, non-capacitationconditions include the absence of one or more of the stimuli needed forcapacitation. Non-capacitated sperm do not undergo acrosome exocytosisinduced by a physiological ligand such as the zona pellucida,solubilized proteins from the zona pellucida, or progesterone. Inaddition, sperm incubated under non-capacitating conditions also willnot demonstrate hyperactivated motility.

In one embodiment, capacitation may be induced in vitro by exposure toexternal stimuli such as bicarbonate and calcium ions, and mediators ofsterol efflux, e.g., 2-hydroxy-propyl-β-cyclodextrin,methyl-β-cyclodextrin, serum albumin, high density lipoprotein,phospholipids vesicles, liposomes, etc. In certain embodiments, anidentifiable change in the G_(M1) localization pattern is observed whensperm are exposed to one or more of these stimuli in vitro.

In one embodiment, after collection, semen samples are typicallyprocessed in some way, including one or more of the following:liquefaction, washing, and/or enrichment. In some embodiments,liquefaction involves allowing the sample to liquefy at room temperatureor at 37° C. (or any temperature there between) for various time periods(typically 15-20 minutes, but ranging from 10-60 minutes). Liquefactionis a process through which the seminal plasma converts from a gel into amore fluid/liquid consistency. Seminal plasma will typically liquefywithout any manipulation, but with especially viscous samples, or ifthere is a desire to hasten the process or make a consistentliquefaction protocol by which all samples are handled, individualsmight manipulate the sample to achieve liquefaction. In certainembodiments the semen sample is manipulated to decrease semen viscosityby using a wide orifice pipette made of non-metallic material. In someembodiments the wide orifice pipette has a gauge size of at least 18gauge, 16 gauge or 14 gauge. In some embodiments, the wide orificepipette has an orifice size of at least 1 mm, 1.2 mm or 1.4 mm. Incertain embodiments, one can also achieve liquefaction by adding variousreagents which do not damage sperm membrane. Reagents which should beavoided are those that damage sperm membrane. The sperm can be washed bycentrifugation and resuspension and subjected to semen analysis, and/orbe subjected to one or more selection processes including: layering ontop of, and centrifugation through a density gradient; layering on topof, and centrifugation through a density gradient followed by collectionof the sperm-enriched fraction followed by resuspension and washing;layering on top of, and centrifugation through a density gradientfollowed by collection of the sperm-enriched fraction and overlaying ontop of that a less dense medium into which motile sperm will swim up; oroverlaying a less dense medium on top of the sample and allowing motilesperm to swim up into it.

After initial processing, the sperm can be counted, and a given numberof sperm can then be placed into containers (such as tubes) containingin vitro non-capacitating or in vitro capacitating medium to achievedesired final concentrations. In one embodiment, the final typicalconcentration of sperm is 1,000,000/ml (final concentration ranges mightvary from 250,000/ml-250,000,000/ml).

The base medium for incubating the sperm under in vitro non-capacitatingand capacitating in vitro conditions can be a physiological bufferedsolution such as, but not limited to, human tubal fluid (HTF); modifiedhuman tubal fluid (mHTF); Whitten's medium; modified Whitten's medium;KSOM; phosphate-buffered saline; HEPES-buffered saline; Tris-bufferedsaline; Ham's F-10; Tyrode's medium; modified Tyrode's medium; TES-Tris(TEST)-yolk buffer; or Biggers, Whitten and Whittingham (BWW) medium.The base medium can have one or more defined or complex sources ofprotein and other factors added to it, including fetal cord serumultrafiltrate, Plasmanate, egg yolk, skim milk, albumin, lipoproteins,or fatty acid binding proteins, either to promote viability or atconcentrations sufficient to help induce capacitation. Typical stimulifor capacitation include one or more of the following: bicarbonate(typically at 20-25 mM, with ranges from 5-50 mM), calcium (typically at1-2 mM, with ranges from 0.1-10 mM), and/or cyclodextrin (typically at1-3 mM, with ranges from 0.1-20 mM). Cyclodextrins may comprise2-hydroxy-propyl-β-cyclodextrin and/or methyl-β-cyclodextrin. Incubationtemperatures are typically 37° C. (ranging from 30° C.−38° C.), andincubation times are typically 1-4 hours (ranging from 30 minutes to 18hours), though baseline samples can be taken at the start of theincubation period (“time zero”).

In one embodiment, for generating patterns of G_(M1), the sperm arewashed with a standard base medium (e.g., phosphate-buffered saline,Modified Whitten's medium, or other similar media) and incubated with alabeling molecule for G_(M1) which has a detectable label on it. SinceG_(M1) has extracellular sugar residues which can serve as an epitope,it can be visualized without having to fix and permeabilize the cells.However, fixation of the cells results in better preservation of thespecimen, easier visualization (compared to discerning patterns inswimming sperm) and allows longer visualization time, while contributingto pattern formation. Various fixatives known for histological study ofspermatozoa are within the purview of those skilled in the art. Suitablefixatives include paraformaldehyde, glutaraldehyde, Bouin's fixative,and fixatives comprising sodium cacodylate, calcium chloride, picricacid, tannic acid and like. In one embodiment, paraformaldehyde,glutaraldehyde or combinations thereof are used.

Fixation conditions can range from about 0.004% (weight/volume)paraformaldehyde to about 4% (weight/volume) paraformaldehyde, althoughabout 0.01% to about 1% (weight/volume) paraformaldehyde is typicallyused. In one embodiment, about 0.005% (weight/volume) paraformaldehydeto about 1% (weight/volume) paraformaldehyde can be used. In oneembodiment, about 4% paraformaldehyde (weight/volume), about 0.1%glutaraldehyde (weight/volume) and about 5 mM CaCl₂ in phosphatebuffered saline can be used.

The period of time a sperm sample is fixed in a fixative may vary. Inone embodiment, a sperm sample is fixed in fixative for about 5 hours orless. In one embodiment, a sperm sample is fixed in a fixative forgreater than about 5 hours. In another embodiment, a sperm sample isfixed in a fixative for about 0.5 hours, for about 1 hours, for about1.5 hours, for about 2 hours, for about 2.5 hours, for about 3 hours,about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours,about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours,about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours,about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours,about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours,about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours,about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours,about 22 hours, about 22.5 hours, about 23 hours, about 23.5 hours,about 24 hours, about 24.5 hours, about 25 hours, about 25.5 hours,about 26 hours, about 26.5 hours, about 27 hours, about 27.5 hours,about 28 hours, about 28.5 hours, about 29 hours, about 29.5 hours,about 30, or any range determinable from the preceding times (forexample, about 26 hours to about 28 hours, or about 3 hours to about 5hours).

The localization pattern of G_(M1) in live or fixed sperm can beobtained by using labeling binding techniques. A molecule havingspecific affinity for the G_(M1) ganglioside can be used. The labelingmolecule can be directly linked to a detectable label (such as afluorophore) or may be detected by a second labeling molecule which hasa detectable label on it. For example, the b subunit of cholera toxin isknown to specifically bind to G_(M1). Therefore, a labeled (such asfluorescent labeled) cholera toxin b subunit can be used to obtain apattern of distribution of G_(M1). In one embodiment, finalconcentrations of the b subunit of cholera toxin linked to fluorophoreare about 10 μg/ml to about 15 μg/ml. In another embodiment, the finalconcentrations of the b subunit of cholera toxin linked to fluorophoreare about 0.1 μg/ml to about 50 μg/ml. Alternatively, a labeled antibodyto G_(M1) can be used. In yet another alternative, a labeled antibody tothe cholera toxin b subunit can be used to visualize the pattern ofG_(M1) staining. And in yet another alternative, a labeled secondaryantibody which binds to either the primary antibody that binds directlyto G_(M1) or to the primary antibody that binds to the b subunit ofcholera toxin could be used. The term “G_(M1) staining” or “staining ofG_(M1)” or “labeling” or related terms as used herein means the stainingseen on or in cells due to the binding of labeled affinity molecules toG_(M1). For example, when fluorescent tagged/labeled cholera toxin bsubunit is used for localization of G_(M1), the signal or staining isfrom the cholera toxin b subunit but is indicative of the location ofG_(M1). The terms “signal” and “staining” and “labeling” are usedinterchangeably. The detectable label is such that it is capable ofproducing a detectable signal. Such labels include a radionuclide, anenzyme, a fluorescent agent or a chromophore. Labeling (or staining) andvisualization of G_(M1) distribution in sperm is carried out by standardtechniques. Labeling molecules other than the b subunit of cholera toxincan also be used. These include polyclonal and monoclonal antibodies.Specific antibodies to G_(M1) ganglioside can be generated by routineimmunization protocols, or can be purchased commercially (e.g., Matreya,Inc., State College, Pa.). The antibodies may be raised against G_(M1)or, can be generated by using peptide mimics of relevant epitopes of theG_(M1) molecule. Identification and generation of peptide mimics is wellknown to those skilled in the art. In addition, the binding of the bsubunit to cholera toxin might be mimicked by a small molecule.Identification of small molecules that have similar binding propertiesto a given reagent is well known to those skilled in the art.

For human sperm, eight different localization patterns (see detailsunder Example 1) were observed when the sperm was under in vitrocapacitating conditions. These patterns are designated as INTER, APM,AA, PAPM, AA/PA, ES, DIFF, and Lined Cell. The INTER, APM, AA, PAPM,AA/PA, ES, and DIFF patterns are shown in FIG. 1 and the Lined-Cellpattern is shown in FIGS. 6A, 6B, 6C, and 6D, each of which are furtherdescribed below:

INTER: The vast majority of the fluorescence is in a band around theequatorial segment, with some signal in the plasma membrane overlyingthe acrosome. There is usually a gradient of signal, with the most atthe equatorial segment and then progressively less toward the tip. Thereis often an increase in signal intensity on the edges of the sperm headin the band across the equatorial segment.

APM (Acrosomal Plasma Membrane): Compared to INTER there is lessdistinction in this pattern between the equatorial signal and thatmoving toward the apical tip. That is, the signal in the plasma membraneoverlying the acrosome is more evenly distributed. APM signal is seeneither from the bright equatorial INTER band moving apically toward thetip, or it can start further up toward the tip and be found in a smallerregion, as it is a continuum with the AA.

AA (Apical Acrosome): In this pattern, the fluorescence is becoming moreand more concentrated toward the apical tip, increased in brightness andreduced in area with signal.

PAPM (Post Acrosomal Plasma Membrane): Signal is exclusively in thepost-acrosomal plasma membrane.

AA/PA (Apical Acrosome/Post Acrosome): Signal is both in the plasmamembrane overlying the acrosome and post-acrosomal plasma membrane.Signal is missing from the equatorial segment.

ES (Equatorial Segment): Bright signal is seen solely in the equatorialsegment. It may be accompanied by thickening of the sperm head acrossthe equatorial region.

DIFF (Diffuse): Diffuse signal is seen over the whole sperm head.

Lined-Cell: Signal is seen at the top of the post-acrosomal region andat the plasma membrane overlying the acrosome as well as the bottom ofthe equatorial segment (i.e., the post acrosome/equatorial band). Signalis missing around the equatorial segment.

The term “G_(M1) localization pattern” is used interchangeably with“pattern” or “localization pattern.”

FIGS. 6A, 6B, 6C, and 6D show Lined-Cell G_(M1) localization patterns ofG_(M1) in sperm from infertile males under capacitating conditions.Specifically, FIG. 6A shows a Lined-Cell G_(M1) localization patternwhere the signal is evenly distributed at the post acrosome/equatorialband and at the plasma membrane overlying the acrosome. FIG. 6B shows aLined-Cell G_(M1) localization pattern where the signal at the plasmamembrane overlying the acrosome is brighter than the signal at the postacrosome/equatorial band. FIGS. 6C and 6D show a signal at the postacrosome/equatorial band that is brighter than the signal at the plasmamembrane overlying the acrosome.

It was observed that while the INTER, AA, APM patterns, and combinationsof these patterns, correlate positively with viable sperm with normalsperm membrane architecture and therefore fertility, the PAPM, AA/PA,ES, DIFF, and the Lined-Cell patterns do not positively correlate withviability, normal membrane architecture and fertility. If incubatedunder non-capacitating conditions, the majority of viable sperm withnormal membrane architecture will exhibit the INTER pattern, which ischaracterized by the majority of labeling being near the equatorialsegment, with the rest extending through the plasma membrane overlyingthe acrosome. Additionally, there is an increase in the number of theAPM and AA patterns upon exposure to stimuli for capacitation. The APMpattern shows more uniform signal in the plasma membrane overlying theacrosome, whereas the AA pattern shows increasing intensity of signal inthe rostral part of the sperm head, the apical acrosome, and reducedsignal moving caudally toward the equatorial segment. Sperm incubatedunder in vitro non-capacitated conditions for infertile individuals haveG_(M1) localization patterns that are similar to G_(M1) localizationpatterns of sperm incubated under in vitro non-capacitated conditionsfor normal individuals.

In one embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps ofexposing a first portion of a sperm sample from a male tonon-capacitating conditions to obtain an in vitro non-capacitated spermsample; exposing a second portion of the sperm sample to capacitatingconditions to obtain an in vitro capacitated sperm sample; fixing the invitro non-capacitated sperm sample and the in vitro capacitated spermsample with a fixative for a time period of at least: one hour, twohours, four hours, eight hours, twelve hours, eighteen hours or twentyfour hours, treating the fixed in vitro non-capacitated sperm sample andthe fixed in vitro capacitated sperm sample with a labeling molecule forG_(M1) localization patterns, wherein the labeling molecule has adetectable label; identifying more than one labeled G_(M1) localizationpatterns for the labeled fixed in vitro non-capacitated sperm sample andthe labeled fixed in vitro capacitated sperm sample, said G_(M1) labeledlocalization patterns being an apical acrosome (AA) G_(M1) localizationpattern, an acrosomal plasma membrane (APM) G_(M1) localization pattern,a Lined-Cell G_(M1) localization pattern and all other labeled G_(M1)localization patterns; comparing the labeled G_(M1) localizationpatterns for the labeled fixed in vitro non-capacitated sperm sample tothe labeled G_(M1) localization patterns for the labeled fixed in vitrocapacitated sperm sample; based on the comparison, assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male based on the identifiedG_(M1) labeled localization patterns for the labeled fixed in vitronon-capacitated sperm sample and the labeled fixed in vitro capacitatedsperm sample. In one embodiment, the characterizing step comprises thesteps of: determining a fertility threshold associated with a percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] for the labeled fixed invitro capacitated sperm sample; wherein a reference percentage of [(AAG_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns], based on distributionstatistics of a known fertile population corresponding to: greater thana percentage that is one standard deviation below the reference meanpercentage indicates fertile; less than a percentage that is onestandard deviation below the reference mean percentage and greater thana percentage that is two standard deviations below the reference meanpercentage indicates sub-fertile; less than a percentage that is twostandard deviations below the reference mean percentage indicatesinfertile; comparing the percentage of [(AA G_(M1) localization patternsplus APM G_(M1) localization patterns)/total G_(M1) localizationpatterns] for the labeled fixed in vitro capacitated sperm sample to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] andidentifying the fertility threshold based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In one embodiment, prior to the exposing steps, a semen sample istreated to decrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembrane chosen from the various reagents that are used to decreasesemen viscosity. In some embodiments, the membrane damaging reagent isselected from the group consisting of (i) a protease; (ii) a nuclease(iii) a mucolytic agent; (iv) a lipase; (v) an esterase and (vi)glycoside hydrolases. In some embodiments, the identifying step isrepeated until the number of Lined-Cell G_(M1) localization patterns issubstantially constant. In one such embodiment, after the identifyingstep is performed, determining the number of Lined-Cell G_(M1)localization patterns, for the labeled fixed in vitro capacitated spermuntil the number is less than 5%, less than 3% of the total number oflabeled cells; or ranges from 1% to 5%, 2 to 5% of the total number oflabeled cells. In another such embodiment, after the identifying step isperformed, determining the number of Lined-Cell G_(M1) localizationpatterns, for the labeled fixed in vitro non-capacitated sperm until thenumber is less than: 25%, 20%, 15% or 10% of the total number of labeledcells; or ranges from 2% to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5%of the total number of labeled cells. In some embodiments the wideorifice pipette has a gauge size of at least 18 gauge, 16 gauge or 14gauge. In some embodiments, the wide orifice pipette has an orifice sizeof at least 1 mm, 1.2 mm or 1.4 mm.

In one such embodiment, the characterizing step may include the stepsof: determining the number of each G_(M1) labeled localization patternsfor a predetermined number of the labeled fixed in vitro non-capacitatedsperm sample; determining the number of each G_(M1) labeled localizationpatterns for a predetermined number of the labeled fixed in vitrocapacitated sperm sample; calculating a ratio for a sum of the number ofAA G_(M1) localization patterns and number of APM G_(M1) localizationpatterns over a sum of the total number of G_(M1) labeled localizationpatterns for the labeled fixed in vitro non-capacitated sperm sample;and calculating a ratio for a sum of the number of AA G_(M1)localization patterns and number of APM G_(M1) localization patternsover a sum of the total number of G_(M1) labeled localization patternsfor the labeled fixed in vitro capacitated sperm sample.

In one such embodiment, the method may further include the steps of:comparing the ratio for the labeled fixed in vitro non-capacitated spermto a ratio of labeled fixed in vitro non-capacitated sperm having aknown fertility status; and comparing the ratio for the labeled fixed invitro capacitated sperm to a ratio of labeled fixed in vitro capacitatedsperm having a known fertility status.

In one embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:obtaining a first portion of a sperm sample from a male that has beenexposed to in vitro non-capacitating conditions, fixed in a fixative forat least: one hour, two hours, four hours, eight hours, twelve hours,eighteen hours or twenty four hours, and treated with a labelingmolecule for G_(M1) localization patterns, wherein the labeling moleculehas a detectable label; obtaining a second portion of the sperm samplethat has been exposed to in vitro capacitating conditions, fixed in afixative, and treated with the labeling molecule for G_(M1) localizationpatterns; identifying more than one G_(M1) labeled localization patternsfor the labeled fixed in vitro non-capacitated sperm sample and thelabeled fixed in vitro capacitated sperm sample, said G_(M1) labeledlocalization patterns being an apical acrosome (AA) G_(M1) localizationpattern, an acrosomal plasma membrane (APM) G_(M1) localization pattern,a Lined-Cell G_(M1) localization pattern and all other labeled G_(M1)localization patterns; comparing the labeled G_(M1) localizationpatterns for the labeled fixed in vitro non-capacitated sperm sample tothe labeled G_(M1) localization patterns for the labeled fixed in vitrocapacitated sperm sample; based on the comparison, assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male based on the identifiedG_(M1) labeled localization patterns for the labeled fixed in vitronon-capacitated sperm sample and the labeled fixed in vitro capacitatedsperm sample. In one embodiment, the characterizing step comprises thesteps of: determining a fertility threshold associated with a percentageof [(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] for the labeled fixed invitro capacitated sperm sample; wherein a reference percentage of [(AAG_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns], based on distributionstatistics of a known fertile population corresponding to: greater thana percentage that is standard deviation below the reference meanpercentage indicates fertile; less than a percentage that is onestandard deviation below the reference mean percentage and greater thana percentage that is two standard deviations below the reference meanpercentage indicates sub-fertile; less than a percentage that is twostandard deviations below the reference mean percentage indicatesinfertile; comparing the percentage of [(AA G_(M1) localization patternsplus APM G_(M1) localization patterns)/total G_(M1) localizationpatterns] for the labeled fixed in vitro capacitated sperm sample to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] andidentifying the fertility threshold based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In some embodiments, the identifying step is repeated until the numberof Lined-Cell G_(M1) localization patterns is substantially constant. Inone such embodiment, after the identifying step is performed,determining the number of Lined-Cell G_(M1) localization patterns, forthe labeled fixed in vitro capacitated sperm until the number is lessthan 5%, less than 3% of the total number of labeled cells; or rangesfrom 1% to 5%, 2 to 5% of the total number of labeled cells. In anothersuch embodiment, after the identifying step is performed, determiningthe number of Lined-Cell G_(M1) localization patterns, for the labeledfixed in vitro non-capacitated sperm until the number is less than: 25%,20%, 15% or 10% of the total number of labeled cells; or ranges from 2%to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5% of the total number oflabeled cells.

In one embodiment of such method, the method further includes the stepsof: determining the number of each G_(M1) labeled localization patternsfor a predetermined number of the labeled fixed in vitro non-capacitatedsperm sample and the labeled fixed in vitro capacitated sperm sample,and calculating a ratio for a sum of the number of AA G_(M1)localization patterns and number of APM G_(M1) localization patternsover a sum of the total number of G_(M1) localization patterns for eachof the labeled fixed in vitro non-capacitated sperm sample and thelabeled fixed in vitro capacitated sperm sample.

In one such embodiment, the characterizing step may further include thesteps of: comparing the ratio for the labeled fixed in vitro capacitatedsperm sample to ratios of G_(M1) localization patterns of in vitrocapacitated sperm for males having a known fertility status; andcomparing the ratio for the labeled fixed in vitro non-capacitated spermsample to ratios of G_(M1) localization patterns in vitronon-capacitated sperm for males having a known fertility status.

In one embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:exposing, in vitro, a sperm sample from a male to capacitatingconditions; fixing the capacitated sperm sample with a fixative for atleast: one hour, two hours, four hours, eight hours, twelve hours,eighteen hours or twenty four hours, treating the fixed in vitrocapacitated sperm sample with a labeling molecule for G_(M1)localization patterns, wherein the labeling molecule has a detectablelabel; identifying more than one G_(M1) labeled localization patternsfor the labeled fixed in vitro capacitated sperm sample, said G_(M1)labeled localization patterns being an apical acrosome (AA) G_(M1)localization pattern, an acrosomal plasma membrane (APM) G_(M1)localization pattern, a Lined-Cell G_(M1) localization pattern and allother labeled G_(M1) localization patterns; assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male. In one embodiment, thecharacterizing step comprises the steps of: determining a fertilitythreshold associated with a percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] for the labeled fixed in vitro capacitated spermsample; wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesfertile; less than a percentage that is one standard deviation below thereference mean percentage and greater than a percentage that is twostandard deviations below the reference mean percentage indicatessub-fertile; less than a percentage that is two standard deviationsbelow the reference mean percentage indicates infertile; comparing thepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] for thelabeled fixed in vitro capacitated sperm sample to the referencepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] andidentifying the fertility threshold based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than one standard deviation below thereference mean percentage indicates abnormal male fertility. Thepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] for thelabeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In one embodiment, prior to the exposing steps, a semen sample istreated to decrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembrane chosen from the various reagents that are used to decreasesemen viscosity. In some embodiments, the membrane damaging reagent isselected from the group consisting of (i) a protease; (ii) a nuclease(iii) a mucolytic agent; (iv) a lipase; (v) an esterase and (vi)glycoside hydrolases. In some embodiments, the identifying step isrepeated until the number of Lined-Cell G_(M1) localization patterns issubstantially constant. In one such embodiment, after the identifyingstep is performed, determining the number of Lined-Cell G_(M1)localization patterns, for the labeled fixed in vitro capacitated spermuntil the number is less than 5%, less than 3% of the total number oflabeled cells; or ranges from 1% to 5%, 2 to 5% of the total number oflabeled cells. In another such embodiment, after the identifying step isperformed, determining the number of Lined-Cell G_(M1) localizationpatterns, for the labeled fixed in vitro non-capacitated sperm until thenumber is less than: 25%, 20%, 15% or 10% of the total number of labeledcells; or ranges from 2% to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5%of the total number of labeled cells. In some embodiments the wideorifice pipette has a gauge size of at least 18 gauge, 16 gauge or 14gauge. In some embodiments, the wide orifice pipette has an orifice sizeof at least 1 mm, 1.2 mm or 1.4 mm.

In one embodiment of such method, the method may further include thesteps of: comparing the ratio of G_(M1) localization patterns to ratiosof G_(M1) localization patterns for males having a known fertilitystatus. In one embodiment, the known fertility status corresponds tofertile males. In another embodiment, the known fertility statuscorresponds to infertile males. In one such embodiment, the comparingstep includes the steps of: determining the number of each G_(M1)labeled localization patterns for a predetermined number of the labeledfixed in vitro capacitated sperm sample, and calculating a ratio for asum of the number of AA G_(M1) localization patterns and number of APMG_(M1) localization patterns over a sum of the total number of G_(M1)labeled localization patterns.

In one embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:obtaining a first portion of a sperm sample from a male that has beenexposed to in vitro capacitating conditions, fixed in a fixative for atleast: one hour, two hours, four hours, eight hours, twelve hours,eighteen hours or twenty four hours, and stained with a labelingmolecule for G_(M1) localization patterns, wherein the labeling moleculehas a detectable label; identifying more than one G_(M1) labeledlocalization patterns for the labeled fixed in vitro capacitated spermsample, said G_(M1) localization patterns being an apical acrosome (AA)G_(M1) localization pattern, an acrosomal plasma membrane (APM) G_(M1)localization pattern, a Lined-Cell G_(M1) localization pattern and allother labeled G_(M1) localization patterns; assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state andassigning the Lined-Cell G_(M1) localization pattern and all otherlabeled G_(M1) localization patterns to a non-capacitated state; andcharacterizing a fertility status of the male. In one embodiment, thecharacterizing step comprises the steps of: determining a fertilitythreshold associated with a percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] for the labeled fixed in vitro capacitated spermsample; wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesfertile; less than a percentage that is one standard deviation below thereference mean percentage and greater than a percentage that is that istwo standard deviations below the reference mean percentage indicatessub-fertile; less than a percentage that is two standard deviationsbelow the reference mean percentage indicates infertile; comparing thepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] for thelabeled fixed in vitro capacitated sperm sample to the referencepercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] andidentifying the fertility threshold based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormal malefertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In some embodiments, the identifying step is repeated until the numberof Lined-Cell G_(M1) localization patterns is substantially constant. Inone such embodiment, after the identifying step is performed,determining the number of Lined-Cell G_(M1) localization patterns, forthe labeled fixed in vitro capacitated sperm until the number is lessthan 5%, less than 3% of the total number of labeled cells; or rangesfrom 1% to 5%, 2 to 5% of the total number of labeled cells. In anothersuch embodiment, after the identifying step is performed, determiningthe number of Lined-Cell G_(M1) localization patterns, for the labeledfixed in vitro non-capacitated sperm until the number is less than: 25%,20%, 15% or 10% of the total number of labeled cells; or ranges from 2%to 25%; 2% to 20%; 2 to 15%; 2 to 10%; 2 to 5% of the total number oflabeled cells.

In one embodiment of such method, the method may further include thesteps of: comparing the ratio of G_(M1) localization patterns to ratiosof G_(M1) localization patterns for males having a known fertilitystatus. In one embodiment, the known fertility status corresponds tofertile males. In another embodiment, the known fertility statuscorresponds to infertile males. In one such embodiment, the comparingstep includes the steps of: determining the number of each G_(M1)labeled localization patterns for a predetermined number of the labeledfixed in vitro capacitated sperm sample, and calculating a ratio for asum of the number of AA G_(M1) localization patterns and number of APMG_(M1) localization patterns over a sum of the total number of G_(M1)labeled localization patterns.

In one embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method includes the steps of:obtaining a sperm sample, wherein at least a portion of the sperm samplehas been exposed to in vitro capacitating conditions to obtain in vitrocapacitated sperm, has been exposed to a fixative for at least: onehour, two hours, four hours, eight hours, twelve hours, eighteen hoursor twenty four hours, and has been stained for G_(M1), obtaining valuesfor one or more semen parameters of the sperm sample; determining aCap-Score of the labeled fixed in vitro capacitated sperm sample basedon one or more G_(M1) labeled localization patterns, said G_(M1) labeledlocalization patterns being an apical acrosome (AA) G_(M1) localizationpattern, a post-acrosomal plasma membrane (APM) G_(M1) localizationpattern, a Lined-Cell G_(M1) localization pattern and all other labeledG_(M1) localization patterns; and calculating a male fertility index(WI) value of the male based on the determined CAP score and the one ormore obtained semen parameters. In one embodiment, the one or more semenparameters of the sperm sample are selected from the group consisting ofvolume of the original sperm sample, concentration of sperm, motility ofsperm, and morphology of sperm.

An embodiment disclosed herein is a method for determining malefertility status. In one embodiment, the method comprises the followingsteps. A sample of in vitro capacitated sperm cells is treated with afluorescence label. One or more capacitated-fluorescence images isobtained wherein the images display one or more G_(M1) localizationpatterns associated with fluorescence labeled in vitro capacitated spermcells. An apical acrosome (AA) G_(M1) localization pattern and anacrosomal plasma membrane (APM) G_(M1) localization pattern are eachassigned to a capacitated state and a Lined-Cell G_(M1) localizationpattern and all other labeled G_(M1) localization patterns are assignedto a non-capacitated state each displayed in the cap-fluorescenceimages. A number for G_(M1) localization patterns is measured, thepatterns comprising AA G_(M1) localization pattern, APM G_(M1)localization pattern, Lined-Cell G_(M1) localization pattern and allother labeled G_(M1) localization patterns, for the fluorescence labeledin vitro capacitated sperm cells, displayed in thecapacitated-fluorescence images to determine a percentage of [(AA G_(M1)localization patterns plus APM G_(M1) localization patterns)/totalG_(M1) localization patterns]. A fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns] corresponding to: greater than a percentage thatis one standard deviation below a reference mean percentage indicatesfertile; less than a percentage that is one standard deviation below areference mean percentage and greater than a percentage that is twostandard deviations below a reference mean percentage indicatessub-fertile; less than a percentage that is two standard deviationsbelow a reference mean percentage indicates infertile. The percentage of[(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In another embodiment, a fertility threshold associated with apercentage of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] isdetermined, wherein a reference percentage of [(AA G_(M1) localizationpatterns plus APM G_(M1) localization patterns)/total G_(M1)localization patterns], based on distribution statistics of a knownfertile population corresponding to: greater than a percentage that isone standard deviation below the reference mean percentage indicatesnormal male fertility; less than a percentage that is one standarddeviation below the reference mean percentage indicates abnormalfertility. The percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns] forthe labeled fixed in vitro capacitated sperm sample is compared to thereference percentage of [(AA G_(M1) localization patterns plus APMG_(M1) localization patterns)/total G_(M1) localization patterns]. Thefertility threshold is identified based on the comparison.

In one such embodiment, the identifying step is also based on one ormore of the following: patient demographics, reproductive status offemale partner, sperm concentration, total motility, progressivemotility, semen volume, semen pH, semen viscosity and/or spermmorphology and combinations thereof.

In various embodiments of the methods described herein, the sperm cellsare treated in vitro with capacitation conditions for a capacitationtime period of: at least one hour; at least 3 hours; at least 12 hours;at least 18 hours; at least 24 hours; for a capacitation time periodranging between 0.5 hours to 3 hours; 3 hours to 12 hours; 6 hours to 12hours; 3 hours to 24 hours; 12 hours to 24 hours; or 18 hours to 24hours.

In various embodiments of the methods described herein, the in vitrocapacitated sperm cells are treated with a fixative for a fixative timeperiod of: at least 0.5 hour; at least 3 hours; at least 12 hours; atleast 18 hours; at least 24 hours; at least 30 hours; at least 36 hours;or at least 48 hours, for a fixation time period ranging between 0.5hours to 3 hours; 3 hours to 12 hours; 6 hours to 12 hours; 3 hours to18 hours; 6-18 hours; 6-24 hours; 12 hours to 24 hours; 18 hours to 24hours; 18-30 hours; 18-36 hours; 24-30 hours; 24-26 hours; 18-48 hours;24-48 hours; or 36-48 hours.

In various embodiments of the methods described herein, the more thanone of G_(M1) labeled localization patterns comprises AA G_(M1)localization pattern, APM G_(M1) localization pattern, Lined-Cell G_(M1)localization pattern, intermediate (INTER) G_(M1) localization pattern,post acrosomal plasma membrane (PAPM) G_(M1) localization pattern,apical acrosome/post acrosome (AA/PA) G_(M1) localization pattern,equatorial segment (ES) G_(M1) localization pattern, and diffuse (DIFF)G_(M1) localization pattern.

In one embodiment, exposing the first portion of the sperm sample tonon-capacitating conditions and exposing the second portion of the spermsample to capacitating conditions occur concurrently.

The male individual may be a human or a non-human animal. In the case ofa non-human animal, identification of patterns that are correlated withfertility status can be carried out based on the teachings providedherein. Non-human animals include horse, cattle, dog, cat, swine, goat,sheep, deer, rabbit, chicken, turkey, various species of fish andvarious zoological species.

In one embodiment, the method of this disclosure provides a method fordesignating a male as likely infertile comprising obtaining G_(M1)localization patterns (e.g., one or more of Lined-Cell, AA, APM, and allother G_(M1) localization patterns) in the sperm from the individual andfrom a normal control that have been incubated under capacitating andnon-capacitating conditions and optionally fixed, and comparing theG_(M1) localization patterns. In the normal control, a statisticallysignificant change in the percentage of sperm displaying certainlocalization patterns would be observed. If the same change is notobserved in the sperm from the test individual, then the individual isdesignated as having an abnormal fertility status. In one embodiment,the patterns that are informative of normal and abnormal fertilitystatus are localization patterns Lined-Cell, INTER, AA and/or APM. Thus,in a sample from an individual who is known to have a normal fertilitystatus (which may be used as a control), there is a higher percentage ofsperm exhibiting AA and/or APM localization patterns, and a lowerpercentage of sperm exhibiting the Lined-Cell and/or INTER localizationpattern upon exposure to in vitro capacitating conditions when comparedto the sperm being exposed to in vitro non-capacitating conditions. Ifno difference or no significant difference is observed in thepercentages of one or more of these localization patterns upon exposureto in vitro capacitating conditions as compared to when the sperm isexposed to in vitro non-capacitating conditions, then the individual isdesignated as having fertility problems. In a variation of the aboveembodiment, the control may be from an individual known to be infertileor sub-fertile. In this embodiment, if the changes in G_(M1) patternsfrom the test individual upon in vitro capacitation in the Lined-Cell,INTER, AA and/or APM localization patterns are the same as the control,then the individual can be deemed as sub-fertile or infertile.

In yet another variation of the above embodiment, the sample from a testindividual may be evaluated without comparing to a control. If nochange, or no significant change, is observed in the number ofLined-Cell, INTER, AA and/or APM patterns upon exposure to in vitrocapacitating conditions, then the individual may be deemed as abnormaland may be designated for further testing, whereas if changes areobserved such that Lined-Cell and/or INTER is decreased, AA isincreased, and/or APM is increased, then the individual may bedesignated as having normal fertility.

In one embodiment, the method comprises analysis of G_(M1) localizationpatterns to identify number of AA and APM patterns in sperm exposed toin vitro capacitating conditions. The number can be expressed as apercentage of one or more of the G_(M1) distribution patterns relativeto the total. In one embodiment, fertility is predicted based on acomparison of the number of AA and/or APM localization patterns againsta predetermined fertility threshold, for example, the threshold (i.e.,cut-off) level between individuals classified as infertile andsub-fertile, or the threshold level between individuals classified assub-fertile and those classified as fertile.

In other embodiments, fertility thresholds may be determined bystatistical analysis of the patterns found in sperm from a population ofmen, with known fertility. For the purposes of this application, a maleis considered fertile or has normal male fertility if the male has apregnant partner or has fathered a child within three years, usingeither natural conception or three or fewer cycles of intra-uterineinsemination. For the purposes of this application, a male is considersub-fertile if the male has failed to achieve a pregnancy with six totwelve months, without use of contraception, and required more thanthree cycles of intra-uterine insemination to achieve a pregnancy. Forthe purposes of this application, a male is considered infertile, if themale has failed to achieve a pregnancy within one year, without use ofcontraception, and failed to achieve a pregnancy using repeated cyclesof intra-uterine insemination. For the purposes of this application,abnormal male fertility includes sub-fertile and infertile males.

As shown in FIG. 4, 73 semen samples were obtained from 24 men known tobe fertile. Their sperm was incubated with stimuli for capacitation, inthis case 4 mM 2-hydroxy-propyl-βcyclodextrin, fixed with 0.01%paraformaldehyde (final concentration). The percentage of cells havingpatterns indicative of having capacitated (e.g., AA+APM) was assessed.The mean percentage of sperm having the AA and APM patterns was 41%, andtwo standard deviations from the mean was calculated as 27% and 55%.

G_(M1) localization patterns in 14 samples from 14 men seeking medicalevaluation of their fertility status were analyzed. The relativepercentages of sperm having AA+APM localization patterns were comparedagainst the statistical thresholds identified from the population ofknown fertile men (FIG. 5). There were no differences observed in thesamples incubated under baseline (non-stimulating, non-capacitatingconditions). However, 5 of the 14 men produced samples that showed lowpercentages of sperm with AA+APM patterns when incubated with 4 mM2-hydroxy-propyl-β-cyclodextrin. These 5 samples all fell below twostandard deviations from the mean. It is believed that approximately30-50% of couples having difficulty conceiving have a component of malefactor. These data fall within that expected range.

In one aspect, the present disclosure provides kits for determination ofmale fertility status. The kit comprises one or more of the following: apipette having an orifice of sufficient size in diameter to preventshearing of a sperm membrane, agents that can act as stimuli for invitro capacitation, capacitating media, non-capacitating media,fixative, labeling reagents s for determining of G_(M1) localizationpatterns, a diagram illustrating one or more G_(M1) localizationpatterns of capacitated sperm and one of more G_(M1) localizationpatterns of non-capacitated sperm. Such G_(M1) localization patterns ofcapacitated sperm and G_(M1) localization patterns of non-capacitatedsperm are reflective of known fertility status. In such a kitembodiment, the fixative composition should not damage sperm membrane.In such embodiments, the reagent that can damage sperm membranes ischosen from the various reagents that are used to decrease semenviscosity. In some embodiments, the membrane damaging reagent includesone or more of a protease, a nuclease, a mucolytic agent, a lipase, anesterase and glycoside hydrolases. In another kit embodiment, thecapacitating media and non-capacitating media, when applied in vitro tosperm cells, produce G_(M1) localization patterns indicative ofcapacitated sperm and patterns indicative of non-capacitated sperm asreflected in the diagram.

In one embodiment, the kit comprises an agent having 4% cyclodextrin tostimulate capacitation.

In one embodiment, the capacitating media comprises: modified humantubal fluid with added 2-hydroxy-propyl-β-cyclodextrin so as to give a 3mM final concentration; the non-capacitating media comprises modifiedhuman tubal fluid; the fixative is 1% paraformaldehyde; and the reagentfor determining G_(M1) patterns is cholera toxin's b subunit (15 μg/mlfinal concentration). In other embodiments, the final concentration ofparaformaldehyde is 0.01%.

An exemplary kit comprises modified HTF medium with gentamicin bufferedwith HEPES (Irvine Scientific, reference 90126). No difference in G_(M1)localization patterns, viability or sperm recovery, and capacitation wasobserved whether bicarbonate- or HEPES-buffered medium was used.Therefore, bicarbonate buffered media can also be used. Use of theHEPES-buffer enables the assay to be performed in clinics using airincubators or water baths, as opposed to only being compatible with CO₂incubators. Similarly, adding supplemental proteins, whether commercial(HTF-SSS™, Irvine Scientific, or plasmanate), or powdered albumin didnot alter recovery or viability, and favorably enhance capacitationstatus.

The exemplary kit can further comprise cell isolation media (such asEnhance S-Plus Cell Isolation Media, 90% from Vitrolife, reference:15232 ESP-100-90%). The exemplary reagents, consumables and procedureswere demonstrated to yield advantageous labeling of G_(M1) on humansperm.

The exemplary kit can further comprise wide orifice pipette tips (200 μllarge orifice tip, USA scientific, 1011-8400). The exemplary kit canfurther comprise wide orifice transfer pipettes (General PurposeTransfer Pipettes, Standard Bulb reference number: 202-20S. VWR catalognumber 14670-147). In one embodiment, the pipette is non-metallic. Insome embodiments the wide orifice pipette has a gauge size of at least18 gauge, 16 gauge or 14 gauge. In some embodiments, the wide orificepipette has an orifice size of at least 1 mm, 1.2 mm or 1.4 mm.

The exemplary kit can further comprise 1.5 ml tubes (Treatment cap,noncap, CD) (USA Scientific 14159700)—one containing cyclodextrin inpowdered form to stimulate capacitation, and one empty fornoncapacitating conditions of media alone. In some embodiments, it ispossible that the cyclodextrin will be found in a separate tube, towhich medium will be added to make a stock solution, that itself wouldbe added to the capacitating tube.

When isolating sperm from seminal plasma it is common for humanandrology labs to collect sperm using density gradients. The exemplarykit can further comprise density gradient materials and/or instructionsto remove the seminal plasma off the density gradient and then tocollect the pelleted sperm using a fresh transfer pipette.

The exemplary kit can further comprise the fixative (such as 0.1% PFA),and optionally comprises informational forms (such as patientrequisition form), labels and containers/bags/pouches and the likeuseful for shipping, storage or regulatory purposes. For example, thekit can contain a foil pouch, a biohazard bag with absorbent for mailingpatient sample, a re-sealable bag with absorbent, and a foam tube placeholder.

The exemplary kit can further include instructions describing any of themethods disclosed herein.

In another aspect, a method for measuring the fertility of a maleindividual is provided. The G_(M1) localization assay can show whethersperm can capacitate, and therefore become competent to fertilize anegg. As described above, the assay may be scored as percentages of themorphologically normal sperm that have specific patterns of G_(M1)localization in the sperm head. The APM and AA patterns increase assperm respond to stimuli for capacitation. Cut-offs can be used todistinguish the relative fertility of the ejaculates, separating thesemen samples into groups based on male fertility (e.g., distinguishingfertile from sub-fertile from infertile men). However, because spermnumber, motility, and morphology can also influence male fertility, thepresent disclosure provides methods for creating an index of malefertility (the “male fertility index” or “MFI”) that encompassesCap-Score and one or more relevant semen parameters (e.g., number,motility, and morphology, etc.). Cap-Score (also referred to as G_(M1)score) is the number of one or more G_(M1) patterns. For example, aCap-Score can be a number of one or more of Lined-Cell, INTER, AA, andAPM. Different indices can be generated that emphasize specific semenparameters. For example, indexes according to the present disclosureinclude:

Cap-Score×% with progressive motility×absolute number;

Cap-Score×% morphologically normal sperm×absolute number;

Cap-Score×% total motility×absolute number×% morphologically normalsperm; and

other variations or combinations of Cap-Score and these parameters, orother specific parameters including those obtained using CASA (computerassisted sperm analysis), such as: VSL (velocity straight line); STR(straightness); LIN (Linearity); VCL (curvilinear velocity); VAP(velocity average path); % motility; duration of motility; LHA (lateralhead amplitude); WOB (wobble); PROG (progression); and BCF (Beat crossnumber), etc. See, e.g., World Health Organization, “WHO LaboratoryManual for the Examination and Processing of Human Sperm,” (Fifth Ed.2010).

The male fertility index may be embodied as a method for measuring thefertility status of a male individual. A sperm sample is obtained,wherein the sperm sample is from the individual being measured andwherein at least a portion of the sperm sample has been exposed to invitro capacitating conditions, exposed to a fixative, and stained forG_(M1), as described above. The values of one or more semen parametersare obtained for the sperm sample, such as, for example, the volume ofthe original sample from the individual, and/or the concentration,motility, and/or morphology of the sperm of the sample. An MFI isdetermined from the number of one or more G_(M1) patterns (e.g., the“CAP” score) and the one or more obtained semen parameter values. In theexamples used herein, the Cap-Score is the percentage of one or moreG_(M1) patterns under capacitating conditions at three hours, but othervariants of Cap-Scores will be apparent in light of this disclosure(e.g., number at other time intervals, change in number of a G_(M1)pattern in capacitated from non-capacitated, etc.)

In one embodiment, a male fertility index score may be calculated for asample of men according to the following equation: Male FertilityIndex/Fertility Group=a+b₁*x₁+b₂*x₂+ . . . +b_(m)*x_(m) where a is aconstant, b₁ through b_(m) are regression coefficients and x₁ thoughx_(m) are male fertility variables such as Cap-Score, motility,morphology, volume, and concentration. Discriminant function analysismay be used to determine which fertility variables discriminate betweentwo or more naturally occurring groups. For example, to determine if anindividual falls into a fertile, sub-fertile or in-fertile group, datawould be collected for numerous fertility variables that describe spermfunction and semen quality. A Discriminant Analysis may then be used todetermine which variable(s) is/are the best predictors of fertilitygroup and relatively how much each fertility variable should beweighted.

The male fertility index may be generated by a lab that reads the G_(M1)localization assay. The lab may obtain a sperm sample, and a semenanalysis corresponding to the sperm sample, from one or more facility(e.g., fertility clinic, sperm bank, etc.). Semen analysis informationcan be included on a card included with a G_(M1) localization assay kit,sent electronically to the lab, and/or otherwise provided. In anotherexemplary embodiment, the lab obtains the Cap-Score of a sperm sampleand also obtains the semen analysis information for the sperm sample. Inone embodiment, the lab calculates the male fertility index based on theobtained Cap-Score and the obtained semen analysis data.

An exemplary method for identifying fertility status of a male comprisesexposing sperm sample from the individual to in vitro non-capacitatingand in vitro capacitating conditions. The sperm are fixed and apercentage of selected G_(M1) patterns in the fixed sperm is determined.The percentage for different G_(M1) patterns in sperm exposed to invitro non-capacitating and in vitro capacitating conditions is compared.A change in the percentage of one or more selected G_(M1) patterns insperm exposed to in vitro capacitating conditions over sperm exposed toin vitro non-capacitating conditions is indicative of the fertilitystatus of the individual. The selected G_(M1) patterns can beLined-Cell, INTER, AA and/or APM. In one embodiment, the fertilitystatus of the individual is determined by calculating a ratio for a sumof the number of AA G_(M1) localization patterns and number of APMG_(M1) localization patterns over a sum of the total number of G_(M1)labeled localization patterns for the capacitated sperm.

An exemplary method for identifying fertility status of a male comprisesexposing a sperm sample from the individual to in vitro capacitatingconditions. The sperm are fixed and a percentage of selected G_(M1)patterns in the fixed sperm is determined. The percentage for differentG_(M1) patterns is compared to the percentage from a control, whereinthe control sperm sample has been exposed to the same in vitrocapacitating conditions and same fixative. A change in the percentage ofone or more selected G_(M1) patterns relative to the change in thecontrol is indicative of different fertility status of the individualthan the fertility status of the control. The G_(M1) patterns can beLined-Cell, INTER, AA and/or APM. In one embodiment, the fertilitystatus of the individual is determined by calculating a ratio for a sumof the number of AA G_(M1) localization patterns and number of APMG_(M1) localization patterns over a sum of the total number of G_(M1)labeled localization patterns for the capacitated sperm. In oneembodiment, prior to the exposing steps, a semen sample is treated todecrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembrane chosen from the various reagents that are used to decreasesemen viscosity. In some embodiments, the membrane damaging reagent isselected from the group consisting of (i) a protease; (ii) a nuclease(iii) a mucolytic agent; (iv) a lipase; (v) an esterase and (vi)glycoside hydrolases. In some embodiments the wide orifice pipette has agauge size of at least 18 gauge, 16 gauge or 14 gauge. In someembodiments, the wide orifice pipette has an orifice size of at least 1mm, 1.2 mm or 1.4 mm.

In the exemplary method, the control can be a sperm sample from anindividual who is known to have normal fertility status or an individualwho is known to have abnormal fertility status. The control can be avalue obtained from a dataset comprising a plurality of individuals, forexample, a dataset comprising at least 50 individuals.

An exemplary method for identifying fertility status of a male asinfertile, sub-fertile, or fertile, comprises exposing a sperm samplefrom the individual to in vitro capacitating conditions. G_(M1) patternsin the sample are determined. The percentage of one or more G_(M1)patterns is compared to a fertility threshold wherein a percentage lessthan the fertility threshold is indicative of fertility problems. Forexample, a percentage less than the fertility threshold can beindicative of a fertility status of infertile or sub-fertile. The G_(M1)patterns can be Lined-Cell, INTER, AA and/or APM. In one embodiment, thefertility status of the individual is determined by calculating a ratiofor a sum of the number of AA G_(M1) localization patterns and number ofAPM G_(M1) localization patterns over a sum of the total number ofG_(M1) labeled localization patterns for the capacitated sperm. In oneembodiment, prior to the exposing steps, a semen sample is treated todecrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembrane chosen from the various reagents that are used to decreasesemen viscosity. In some embodiments, the membrane damaging reagent isselected from the group consisting of (i) a protease; (ii) a nuclease(iii) a mucolytic agent; (iv) a lipase; (v) an esterase and (vi)glycoside hydrolases. In some embodiments the wide orifice pipette has agauge size of at least 18 gauge, 16 gauge or 14 gauge. In someembodiments, the wide orifice pipette has an orifice size of at least 1mm, 1.2 mm or 1.4 mm.

The in vitro capacitating conditions in the exemplary methods caninclude exposure to i) bicarbonate and calcium ions, and ii) mediatorsof sterol efflux such as 2-hydroxy-propyl-β-cyclodextrin,methyl-β-cyclodextrin, serum albumin, high density lipoprotein,phospholipids vesicles, fetal cord serum ultrafiltrate, fatty acidbinding proteins, or liposomes. In the exemplary methods, exposure ofthe control to capacitating or non-capacitating conditions can be donein parallel with the test sample.

An exemplary method for identifying fertility status of a male asinfertile, sub-fertile, or fertile, comprises exposing a sperm samplefrom the individual to capacitating conditions. The percentage of eachG_(M1) pattern in the sample is determined. The percentage of one ormore G_(M1) patterns is compared to an infertility threshold wherein apercentage less than the infertility threshold is indicative offertility problems. The capacitating conditions in the exemplary methodcan include exposure to i) bicarbonate and calcium ions, and ii)mediators of sterol efflux such as 2-hydroxy-propyl-β-cyclodextrin,methyl-β-cyclodextrin, serum albumin, high density lipoprotein,phospholipids vesicles, fetal cord serum ultrafiltrate, fatty acidbinding proteins, or liposomes. The one or more G_(M1) localizationpatterns can be Lined-Cell, INTER, AA and/or APM. In one embodiment, thefertility status of the individual is determined by calculating a ratiofor a sum of the number of AA G_(M1) localization patterns and number ofAPM G_(M1) localization patterns over a sum of the total number ofG_(M1) labeled localization patterns for the capacitated sperm. In oneembodiment, prior to the exposing steps, a semen sample is treated todecrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembrane chosen from the various reagents that are used to decreasesemen viscosity. In some embodiments, the membrane damaging reagent isselected from the group consisting of (i) a protease; (ii) a nuclease(iii) a mucolytic agent; (iv) a lipase; (v) an esterase and (vi)glycoside hydrolases. In some embodiments the wide orifice pipette has agauge size of at least 18 gauge, 16 gauge or 14 gauge. In someembodiments, the wide orifice pipette has an orifice size of at least 1mm, 1.2 mm or 1.4 mm.

The fertility threshold in the exemplary methods can be the AA+APMpattern percentage at which the fertility of a population ceases tosubstantially increase. For example, the fertility threshold can be alevel of AA+APM at which more than 50% of the population are fertile; alevel of AA+APM at which more than 60-85% of a population is fertile; ora level of AA+APM in the range of 35-40 (relative percentage of totalG_(M1) patterns), inclusive. The fertility threshold can be 38, 38.5,39, or 39.5% AA+APM (relative to total G_(M1) patterns).

An exemplary method may further comprise comparing the percentage of oneor more G_(M1) patterns to an infertility threshold wherein a percentageless than the infertility threshold is indicative of infertility. Forexample, the infertility threshold can be the AA+APM pattern percentageat which the fertility of a population begins to substantially increase;a level of AA+APM at which less than 50% of the population are fertile;a level of AA+APM at which more than 60-85% of a population is fertile;or a level of AA+APM in the range of 14-18 (relative percentage of totalG_(M1) patterns), inclusive. The infertility threshold can be 14, 14.5,15, or 15.5% AA+APM (relative to total G_(M1) patterns).

An exemplary method for identifying fertility status of a male comprisesobtaining sperm samples, wherein the sperm samples are from theindividual and wherein the sperm samples have been exposed tonon-capacitating or capacitating conditions, fixed, and stained forG_(M1). The number of selected G_(M1) patterns in the sperm isdetermined. The percentage for different G_(M1) patterns in spermexposed to in vitro non-capacitating and in vitro capacitatingconditions is compared. A change in the percentage of one or moreselected G_(M1) patterns in sperm exposed to in vitro capacitatingconditions over sperm exposed to in vitro non-capacitating conditions isindicative of the fertility status of the individual. The G_(M1) patterncan be selected from the group consisting of AA, APM, INTER, Lined-Celland combinations thereof. In one embodiment, the fertility status of theindividual is determined by calculating a ratio for a sum of the numberof AA G_(M1) localization patterns and number of APM G_(M1) localizationpatterns over a sum of the total number of G_(M1) labeled localizationpatterns for the capacitated sperm.

An exemplary method for identifying fertility status of a maleindividual comprises obtaining a sperm sample, wherein the sperm sampleis from the individual and wherein the sperm sample has been exposed toin vitro capacitating conditions, has been fixed and has been stainedfor the presence of G_(M1). A number of selected G_(M1) patterns in thesperm is determined. The percentage for one or more different G_(M1)patterns is compared to the percentage of patterns from a control orpredetermined criteria. The control sperm sample has been exposed to thesame in vitro capacitating conditions and same fixative. A change in thepercentage of one or more selected G_(M1) patterns relative to thechange in the control is indicative of different fertility status of theindividual than the fertility status of the control.

An exemplary method for identifying fertility status of a maleindividual comprises obtaining a sperm sample, wherein the sperm sampleis from the individual, and wherein the sperm sample has been exposed toin vitro capacitating conditions, has been fixed, and has been stainedfor G_(M1) patterns. The G_(M1) localization patterns in the sample aredetermined. The percentage of one or more G_(M1) patterns is compared toan infertility threshold wherein a percentage less than the infertilitythreshold is indicative of fertility problems.

An exemplary method for measuring the fertility status of a maleindividual comprises obtaining a sperm sample, wherein the sperm sampleis from the individual, and wherein the sperm sample has been exposed toin vitro capacitating conditions, has been exposed to a fixative, andhas been stained for G_(M1). Values are obtained for one or more ofvolume of the original sample, and concentration, motility, andmorphology of the sperm in the original sample. A Cap-Score of the spermsample is determined as the percentage of one or more G_(M1)localization patterns in the sample. A male fertility index (MFI) valueof the individual is calculated based on the determined Cap-Score andthe one or more obtained volume, concentration, motility, andmorphology. For example, the MFI value can be calculated by multiplyingthe Cap-Score, the volume, the concentration, the motility value, andthe morphology value. The motility can be a percentage of the spermwhich are motile. The morphology can be a percentage of the sperm thatare morphologically normal.

An exemplary method for measuring the fertility status of a maleindividual comprises obtaining a Cap-Score of a sperm sample of theindividual as the percentage of one or more G_(M1) localization patternsin the sample. Values are obtained for one or more of volume of theoriginal sample, and concentration, motility, and morphology of thesperm in the original sample. A male fertility index (MFI) value of theindividual is calculated based on the determined Cap-Score and the oneor more obtained volume, concentration, motility, and morphology.

The invention is further described through the following illustrativeexamples, which are not to be construed as restrictive.

Example 1

This example provides demonstration of G_(M1) localization patternsobtained with human sperm. Ejaculated sperm were collected from maledonors, and allowed to liquefy for 20 mins at 37° C., and then volume,initial count, motility and morphology assessments were performed. 1 mlof the semen sample was layered on top of 1 ml of a density gradient(90% Enhance-S; Vitrolife, San Diego, Calif., USA) in a 15 ml conicaltube. The tube was centrifuged at 300×g for 10 minutes. The bottom 1 mlfraction was transferred to a new 15 ml tube and then resuspended in 4ml of mHTF. This was centrifuged at 600×g for 10 minutes. Thesupernatant was removed and the pellet of sperm was resuspended in 0.5ml of mHTF. The washed sperm were then evaluated for concentration andmotility. Equal volumes of sperm were then added to two tubes, such thatthe final volume of each tube was 300 μl, and the final concentration ofsperm was 1,000,000/ml. The first tube contained mHTF (non-capacitatingcondition) and the second tube contained mHTF plus2-hydroxy-propyl-β-cyclodextrin at a final concentration of 3 mM(capacitating condition). Sperm were incubated for varying lengths oftime, but 3 hours was typically used. These incubations were performedat 37° C.

At the end of the incubation period, the contents of each tube weremixed gently, and 18 μl from each tube was removed and transferred toseparate microcentrifuge tubes. 2 μl of 1% (weight/volume)paraformaldehyde was added to achieve a final concentration of 0.1%. Inanother embodiment, 0.1% (weight/volume) paraformaldehyde was added toachieve a final concentration of 0.01%. These tubes were mixed gentlyand incubated at room temperature for 15 minutes, at which time 0.3 μlof 1 mg/ml cholera toxin b subunit was added. The contents of the twotubes were again mixed gently and allowed to incubate for an additional5 minutes at room temperature. From each tube, 5 μl was removed andplaced on a glass slide for evaluation by fluorescence microscopy. Toprovide a counter-stain, speeding determination of focal planes andincreasing longevity of the fluorescence signal, 3 μl of DAPI/Antifadewas sometimes added.

As shown in FIG. 2, localization patterns of G_(M1) in normal humansperm reflect response to capacitating conditions. Full response is seenonly in men with normal fertility; the responsive pattern was largelyreduced or absent in men with unexplained infertility who have failed onprevious attempts at intrauterine insemination (IUI) or in vitrofertilization (IVF). FIG. 1 shows the G_(M1) patterns in human sperm.However, for the purpose of the diagnostic assay, patterns reflectingabnormalities such as PAPM, AA/PA, ES, and DIFF can be grouped for easeof analysis. FIGS. 2A-2C show the relative distributions of thedifferent patterns in normal semen incubated under non-capacitatingconditions (NC; FIG. 2A), or capacitating conditions (CAP; FIG. 2B). Areduction in INTER pattern is seen in normal semen upon exposure to CAP(FIG. 2C), while significant increases in the AA pattern and the APMpattern are also seen. In comparison with these normal data, sperm froma group of men known to have unexplained infertility were also subjectedto the G_(M1) assay. In these sperm, there was almost no increase in theAA pattern or the APM pattern under capacitating conditions.

Example 2

In this example, clinical histories of 34 patients were studied toperform a close analysis of their G_(M1) assay scores relative tohistory of ever achieving clinical evidence of pregnancy. A male patientwas defined as “fertile” if a patient couple achieved some evidence offertilization/clinical pregnancy (even if limited to biochemicalevidence or a sac without heartbeat on ultrasound) within 3 or fewercycles.

Analysis of the data for these 34 patients revealed that if one applieda cut-off of 40% (APM+AA) for the score of the capacitated samples atthe 3 hour time point, then 7/8 who “passed” (having a score of 39.5% orgreater), were found to have been designated “fertile” (87.5%). Of the26 who “failed” (having a score of 39.4 or less), only 3/26 had evidenceof clinical pregnancy (11.5%). (see Table 1 below).

If one reduces the cutoff, it would be predicted that more people whoare clinically sub-fertile will get a passing score and the percentagethat pass the assay and are fertile within 3 cycles should go down.Interestingly, the result was not a smooth gradient or continuous curvein terms of fertility (as defined by the ≤=3 cycle criterion). That is,whether one failed the assay as defined at 40 or 35 didn't correlatewith any significant change in chance of fertility, which was always low(between 11.5-14.3%). Conversely, passing the assay at 35 vs 40corresponded with a very large difference in chances of fertility(ranging from 53.8-87.5%, respectively). To reinforce and reiterate thispoint, a change in 5% of the combined APM+AA percentages correspondedwith over a 30% change in history of fertility.

These results suggest that male fertility is more like a “stepfunction,” in which ranges of scores for the male fertility assaycorrespond with categorizations of “fertile,” “sub-fertile” or“infertile,” rather than small changes in scores equating withcorrespondingly small but continuous changes in male fertility (chanceof achieving clinical pregnancy). These data indicate strongly that ascore of roughly 38.5-40 would be the cut-off between designations of“sub-fertile” or “fertile.” Further examination of the data suggest thata cut-off of <14.5% could be used as a designation of likely“infertile.”

Cut-Off Fertile Defined on Conceiving Within </=3 cycles 39.5 Pass 8(7/8 fertile = 87.5%) Fail 26 (3/26 fertile = 11.5%) 38.5 Pass 8 (7/8fertile = 87.5%) Fail 26 (3/26 fertile = 11.5%) 37.5 Pass 11 (7/11fertile = 63.6%) Fail 23 (3/23 fertile = 13.0%) 36.5 Pass 11 (7/11fertile = 63.6%) Fail 23 (3/23 fertile = 13.0%) 35.5 Pass 13 (7/13fertile = 53.8%) Fail 21 (3/21 fertile = 14.3%)

Summarizing data for these men, who were all similar in terms of averagesemen parameters, suggest the following ranges (based on absolutescores): Infertile: <14.5, sub-fertile: 14.5-38.4, fertile: ≥38.5.

Alternatively, one can evaluate the fertility of a sample by comparingthe change in relative number of the APM and/or AA patterns over thetime of incubation under capacitating conditions, or against therelative number observed under non-capacitating conditions. For example,one could compare the APM+AA relative number after 3 hours of incubationin capacitating conditions with the relative number of those patterns atthe start of incubation. In yet another embodiment, one might comparethe change in APM and/or AA frequencies with results obtained fromsuccessive time points (such as 1, 2, and 3 hours). In effect, one canplot the relative frequencies on the Y axis and time points on the Xaxis, and evaluate the slope or rate of change of the increasing numberof one or more of the INTER, APM and/or AA samples undernon-capacitating and capacitating conditions. When this approach to theanalysis was performed in a group of 63 patients, 31 men with scoresmatching the normal reference group were identified, with baselineG_(M1) patterns of 17%-22%-28% in non-capacitating and 26%-31%-38% incapacitating media, respectively over 1, 2, and 3 hours of incubation(see FIG. 3). 32 men with below reference values of 15%-20%-24% innon-capacitating and 20%-25%-29% in capacitating media were identified.Semen analysis parameters of number, motility and percent normalmorphology (using strict WHO criteria) were comparable between the twogroups. The population with normal range G_(M1) patterns had anintrauterine insemination (IUI) pregnancy rate of 45.2% (14/31) of which8 (25.8%) generated at least one fetal heartbeat. Three additionalcouples in this group became pregnant on their own. For men withbelow-reference G_(M1) patterns, the IUI clinical pregnancy rate wasonly 6.3% (2/32; P=0.03). In this cohort, 13 underwent ICSI and 6 becamepregnant (46.2%).

Example 3

Sperm cells were treated as described in Example 1 but incubated infixative for 24 hours. The labeled sperm cells were then evaluated byfluorescence microscopy. A new G_(M1) localization pattern, Lined-Cellswas identified as illustrated in FIGS. 6A, 6B, 6C and 6D. InLined-cells, as illustrated in FIG. 6A, there is G_(M1) signal at thebottom of the equatorial segment/top of the post acrosomal region, andat the plasma membrane overlying the acrosome. The signal is evenlydistributed in the post acrosome/equatorial region and the plasmamembrane overlying the acrosome. There is also a band at the equatorialsegment that lacks signal. As illustrate in FIG. 6B, the signal at theplasma membrane overlying the acrosome is brighter than the signal atthe post acrosome/equatorial band. As illustrated in FIGS. 6C and 6D,the signal found at the post acrosome/equatorial band is brighter thanthe signal at the plasma membrane overlying the acrosome.

Sperm cells from a single donor were washed, incubated under bothcapacitating (Stim) and non-capacitating (Non-Stim) conditions and thenscored both on day 0 (maintained in fix for approximately 5 hours) andday 1 (maintained in fix for approximately 27.5 hours). There was littlechange in the percentage of Lined-cells from day 0 to day 1 for the Stimtreatment. In contrast, the percent of lined cells from day 0 to day 1increased from 3 to 22% for the Non-Stim treatment. In conjunction withthis change, there was a subsequent decrease in INTER from 76 to 51%.These data are consistent with lined cell patterns developing on day 1from cells having an inter pattern on day 0.

TABLE 1 % AA % APM % Inter % Lined Cells % Other Non-Stim day 0 0 16 763 6 Stim day 0 7 19 62 4 8 Non-Stim day 1 4 14 51 22 10 Stim day 1 4 2453 3 16

Example 4

Cells from a single donor were washed, incubated under both capacitating(Stim) and non-capacitating (Non-Stim) conditions and then scored bothon day 0 (maintained in fix for approximately 4 hour) and day 1(maintained in fix for approximately 25 hours). Since few lined cellswere observed on day 0, similar Cap-Scores were obtained on day 0 withor without including the number of Lined-cells for determining theCap-Scores. However, with the emergence of Lined-cells on day 1,different Cap-Score™ values could be obtained depending on how theLined-cells were interpreted. For example, when Lined-cells were treatedas Non-Capacitated, similar Cap-Scores™ were obtained for both the Stimand non-Stim treatments. However, if the Lined-cells were treated ascapacitated, separated or removed from the Cap-Score™ calculation,greater Cap-Scores were obtained for the Non-Stim treatment than for theStim treatment. Having larger Cap-Score™ values for the Non-Stimtreatment makes no sense, as these sperm cells were incubated underbasal conditions and thus would not have shown G_(M1) patternsassociated with capacitation. These observations provide furthercomplementary evidence that Lined-cells represent a non-capacitatedstate and should be treated as such when calculating Cap-Score™.

TABLE 2 Cap-Score ™ computed with: Lined cells Lined Lined Lined as Non-cells as cells Cells Capacitated Capacitated separated removed Non-Stimday 0 18 20 20 19 Stim day 0 28 31 30 29 Non-Stim day 1 35 52 52 42 Stimday 1 36 39 38 37

Example 5

Forty different semen samples, from 18 unique donors were washed, thesperm were incubated under both capacitating (Stim) and non-capacitating(Non-Stim) conditions and then scored both on day 0 and day 1. Asignificant correlation is observed between Stim Cap-Score™ valuesobtained on day 0 and day 1 when Lined-cells are treated asnon-capacitated (r=0.32; n=40; p<0.05). In contrast, no correlation isobserved between day 0 and day 1 when Lined-cells are treated ascapacitated, separated into either capacitated or non-capacitated binsbased on G_(M1) localization pattern, or simply removed from theCap-Score™. These observations support the view that the Lined-cellslocalization pattern develops as sperm are maintained overnight and thaton day 0 these sperm exhibit an inter/non-capacitated pattern. Thetreatment of Lined-cells as non-capacitated, stabilizes the Cap-Score™over time and is consistent with this pattern reflecting cells that areinfertile. What's more, these data demonstrate that interpretation ofthe Lined-cells as non-capacitated is applicable to the population.Nonetheless, the appearance of Lined-cells on day 1 is donor dependent.This raises the possibility that observation of these Lined-cells mayprovide additional information about these donors and the ability oftheir sperm to fertilize.

Although the present disclosure has been described with respect to oneor more particular embodiments, it will be understood that otherembodiments of the present disclosure may be made without departing fromthe spirit and scope of the present disclosure, and such otherembodiments are intended to be within the scope of this disclosure.

1.-34. (canceled)
 35. A method comprising: exposing, in vitro, a spermsample from a male to capacitating conditions; fixing the capacitatedsperm sample with a fixative for at least 0.5 hour; treating the fixedin vitro capacitated sperm sample with a labeling molecule for G_(M1)localization patterns, wherein the labeling molecule has a detectablelabel; identifying more than one G_(M1) labeled localization patternsfor the labeled fixed in vitro capacitated sperm sample, said G_(M1)labeled localization patterns being an apical acrosome (AA) G_(M1)localization pattern, an acrosomal plasma membrane (APM) G_(M1)localization pattern, a Lined-Cell G_(M1) localization pattern, a postacrosomal plasma membrane (PAMP) G_(M1) localization pattern, an apicalacrosome/post acrosome (AA/PA) G_(M1) localization pattern, anintermediate (INTER) G_(M1) localization pattern, an equatorial segment(ES) G_(M1) localization pattern, and a diffuse (DIFF) G_(M1)localization pattern; assigning the apical acrosome (AA) G_(M1)localization pattern and the acrosomal plasma membrane (APM) G_(M1)localization pattern to a capacitated state; determining a fertilitythreshold corresponding to a ratio of (i) the number of G_(M1) labeledlocalization patterns assigned to the capacitated state and (ii) a totalnumber of G_(M1) labeled localization patterns; comparing the fertilitythreshold to a reference value; determining a fertility status of themale based on said comparison; and identifying an appropriate assistedreproductive technology.
 36. The method of claim 35, wherein thefertility threshold is a percentage of [(AA G_(M1) localization patternsplus APM G_(M1) localization patterns)/total G_(M1) localizationpatterns] for the labeled fixed in vitro capacitated sperm sample; andthe reference value of [(AA G_(M1) localization patterns plus APM G_(M1)localization patterns)/total G_(M1) localization patterns] is based ondistribution statistics of a known fertile population wherein afertility threshold that is (i) greater than a percentage that is onestandard deviation below the reference value indicates fertile; (ii)less than a percentage that is one standard deviation below thereference mean percentagevalue and greater than a percentage that is twostandard deviations below a reference value indicates sub-fertile; and(iii) less than a percentage that is two standard deviations below thereference value indicates infertile.
 37. The method of claim 35, furthercomprising the step of: comparing a ratio of G_(M1) localizationpatterns to ratios of G_(M1) localization patterns for males having aknown fertility status.
 38. The method of claim 37, wherein thecomparing step comprises: determining the number of each G_(M1) labeledlocalization patterns for a predetermined number of the labeled fixed invitro capacitated sperm sample, and calculating a ratio for a sum of thenumber of AA G_(M1) localization patterns and number of APM G_(M1)localization patterns over a sum of the total number of G_(M1) labeledlocalization patterns.
 39. (canceled)
 40. The method of claim 35,wherein the capacitating conditions include exposure to one or more ofbicarbonate ions, calcium ions, and a mediator of sterol efflux.
 41. Themethod of claim 40, wherein the mediator of sterol efflux is2-hydroxy-propyl-β-cyclodextrin, methyl-β-cyclodextrin, serum albumin,high density lipoprotein, phospholipid vesicles, fetal cord serumultrafiltrate, fatty acid binding proteins, or liposomes.
 42. The methodof claim 41, wherein the mediator of sterol efflux is2-hydroxy-propyl-β-cyclodextrin.
 43. The method of claim 37, wherein theknown fertility status corresponds to fertile males.
 44. The method ofclaim 37, wherein known fertility status corresponds to infertile males.45. The method of claim 35, wherein the fixative comprisesparaformaldehyde, glutaraldehyde or combinations thereof.
 46. The methodof claim 35, wherein the labeling molecule for G_(M1) localizationpatterns is fluorescent labeled cholera toxin b subunit.
 47. The methodof claim 35, wherein the identifying step is performed from 2 to 24hours after the exposing step.
 48. The method of claim 35, furthercomprising the step of: prior to the exposing step, treating a semensample to decrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembranes.
 49. A method comprising: obtaining a first portion of a spermsample from a male that has been exposed to in vitro capacitatingconditions, fixed in a fixative for at least 0.5 hour, and stained witha labeling molecule for G_(M1) localization patterns, wherein thelabeling molecule has a detectable label; identifying more than oneG_(M1) labeled localization patterns for the labeled fixed in vitrocapacitated sperm sample, said G_(M1) localization patterns being anapical acrosome (AA) G_(M1) localization pattern, an acrosomal plasmamembrane (APM) G_(M1) localization pattern, a Lined-Cell G_(M1)localization pattern, a post acrosomal plasma membrane (PAMP) G_(M1)localization pattern, an apical acrosome/post acrosome (AA/PA) G_(M1)localization pattern, an intermediate (INTER) G_(M1) localizationpattern, an equatorial segment (ES) G_(M1) localization pattern, and adiffuse (DIFF) G_(M1) localization pattern; assigning the apicalacrosome (AA) G_(M1) localization pattern and the acrosomal plasmamembrane (APM) G_(M1) localization pattern to a capacitated state;determining a fertility threshold corresponding to a ratio of (i) thenumber of G_(M1) labeled localization patterns assigned to thecapacitated state and (ii) a total number of G_(M1) labeled localizationpatterns; comparing the fertility threshold to a reference value;determining a fertility status of the male based on said comparison; andidentifying an appropriate assisted reproduction technology.
 50. Themethod of claim 49, wherein the fertility threshold is a percentage of[(AA G_(M1) localization patterns plus APM G_(M1) localizationpatterns)/total G_(M1) localization patterns] for the labeled fixed invitro capacitated sperm sample; wherein a reference value of [(AA G_(M1)localization patterns plus APM G_(M1) localization patterns)/totalG_(M1) localization patterns] is based on distribution statistics of aknown fertile population wherein a fertility threshold that is (i)greater than a percentage that is one standard deviation below thereference mean percentage indicates fertile; (ii) less than a percentagethat is one standard deviation below the reference mean percentage andgreater than a percentage that is two standard deviations below areference mean percentage indicates sub-fertile; and (iii) less than apercentage that is two standard deviations below the reference meanpercentage indicates infertile.
 51. The method of claim 49, furthercomprising the step of: comparing a ratio of G_(M1) localizationpatterns to ratios of G_(M1) localization patterns for males having aknown fertility status.
 52. The method of claim 51, wherein thecomparing step comprises: determining the number of each G_(M1) labeledlocalization patterns for a predetermined number of the labeled fixedcapacitated sperm sample, and calculating a ratio for a sum of thenumber of AA G_(M1) localization patterns and number of APM G_(M1)localization patterns over a sum of the total number of G_(M1) labeledlocalization patterns.
 53. (canceled)
 54. The method of claim 49,wherein the capacitating conditions include exposure to one or more ofbicarbonate ions, calcium ions, and a mediator of sterol efflux.
 55. Themethod of claim 54, wherein the mediator of sterol efflux is2-hydroxy-propyl-β-cyclodextrin, methyl-β-cyclodextrin, serum albumin,high density lipoprotein, phospholipid vesicles, fetal cord serumultrafiltrate, fatty acid binding proteins, or liposomes.
 56. The methodof claim 55, wherein the mediator of sterol efflux is2-hydroxy-propyl-β-cyclodextrin.
 57. The method of claim 51, wherein theknown fertility status corresponds to fertile males.
 58. The method ofclaim 51, wherein the known fertility status corresponds to infertilemales.
 59. The method of claim 49, wherein the fixative comprisesparaformaldehyde, glutaraldehyde or combinations thereof.
 60. The methodof claim 49, wherein the labeling molecule for G_(M1) localizationpatterns is fluorescent labeled cholera toxin b subunit.
 61. The methodof claim 49, wherein the identifying step is performed from 2 to 24hours after the exposing step.
 62. The method of claim 49, furthercomprising the step of: prior to the obtaining step, treating a semensample to decrease semen viscosity using a wide orifice pipette made ofnon-metallic material and using a reagent that does not damage spermmembranes. 63.-71. (canceled)